KR102815803B1 - Antibodies specific for glycated BTLA (B- and T-lymphocyte attenuating factor) - Google Patents

Abstract

The present invention provides molecules, such as antibodies, that selectively bind to glycosylated BTLA (B- and T-lymphocyte attenuating factor) over non-glycosylated BTLA. Methods of making and using such molecules, including methods of treating or diagnosing cancer, are also provided. In some embodiments, the anti-glycosylated BTLA antibodies provided herein can immunospecifically bind to glycosylated wild-type BTLA (WT). In some embodiments, the anti-glycosylated BTLA antibodies provided herein can immunospecifically bind to one or more BTLA double mutants that have only a single glycosylation site at BTLA N75, N94 or N110. In some embodiments, the anti-glycosylated BTLA antibodies provided herein exhibit only background binding, if any, to BTLA triple mutants that do not have a glycosylation site at any of BTLA N75, N94 or N110.

Description

Antibodies specific for glycated BTLA (B- and T-lymphocyte attenuating factor)

1. Cross-reference to related applications

This application claims the benefit of priority to U.S. Provisional Application No. 62/262,293, filed December 2, 2015, which is incorporated herein by reference in its entirety.

2. Sequence list

This application contains a sequence listing which is being filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 30, 2016, is named 604556-228009_SL.txt and is 48,346 bytes in size.

3. Technical field

The present invention relates generally to the fields of medicine, molecular biology and oncology. More specifically, the present invention relates to antibodies for the treatment of cancer.

4. Background Technology

The immune system of humans and other mammals protects them from infection and disease. Upregulation of co-inhibitory molecules by tumor cells or tumor-infiltrating lymphocytes appears to be a mechanism by which tumors evade immune responses, dampening T-cell responses to cancer. Today, a variety of co-inhibitory molecules, including T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), have been implicated in immune evasion of cancer cells. Antagonistic antibodies have been developed to overcome immune evasion, and anti-CTLA-4 and anti-PD-1 antibodies have been tested in clinical trials with encouraging results. However, there is an urgent need for the development of novel therapeutics that safely and effectively treat diseases by modulating the immune system. The compositions and methods disclosed herein meet this need and provide other related advantages.

5. Summary of the invention

The present invention provides isolated monoclonal antibodies that selectively bind to glycosylated B- and T-lymphocyte attenuator ("BTLA") over non-glycosylated BTLA. In some embodiments, the antibodies selectively bind to BTLA that is glycosylated at positions N75, N94, and/or N110 over non-glycosylated BTLA.

In some embodiments, the isolated antibody provided herein selectively binds human BTLA that is glycosylated at N75, N94, N110 or any combination thereof, as compared to non-glycosylated BTLA. In some embodiments, the isolated antibody selectively binds human BTLA having N75 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N94 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N110 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N75 and N94 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N94 and N110 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N75 and N110 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N75, N94 and N110 glycosylation.

In some embodiments, the antibody selectively binds to one or more glycosylation motifs. In some embodiments, the antibody binds to a glycopeptide comprising a glycosylation motif and an adjacent peptide. In some embodiments, the antibody binds to a peptide sequence located proximal to one or more of the glycosylation motifs in three dimensions. In some embodiments, the antibody binds to glycosylated BTLA with a Kd that is less than half the Kd exhibited for non-glycosylated BTLA. In a further embodiment, the antibody binds to glycosylated BTLA with a Kd that is at least 10-fold less than the Kd exhibited for non-glycosylated BTLA.

In some embodiments, the antibody specifically masks a glycosylation motif of BTLA comprising BTLA positions N75, N94, N110, or any combination thereof.

In some embodiments, binding of the antibody to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than the fluorescence intensity exhibited by non-glycosylated BTLA.

In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32.

In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.

In some embodiments, the antibody comprises (a)(1) a V H CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 6, 34, or 62, (ii) SEQ ID NO: 9, 37, or 65, (iii) SEQ ID NO: 12, 40, or 68, and (iv) SEQ ID NO: 15, 43, or 71; (2) a V _H CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 7, 35, or 63, (ii) SEQ ID NO: 10, 38, or 66, (iii) SEQ ID NO: 13, 41, or 69, and (iv) SEQ ID _NO : 16, 44, or 72; and (3)(i) a heavy chain variable (V H ) region comprising a V H CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 36, or 64, (ii) SEQ ID NO: 11, 39, or 67, (iii) SEQ ID NO: 14, 42, or 70, and (iv) SEQ ID NO: 17, 45, or 73; and/or ( _b )(1) a V _L CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 18, 46, or 74, (ii) SEQ ID NO: 21, 49, or 77, (iii) SEQ ID NO: 24, 52, or 80, and ( _iv ) SEQ ID NO: 27, 55, or 83; (2) a light chain variable (V L ) region comprising a V L CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 19, 47, or 75, (ii) SEQ ID NO: 22, 50, or 78, (iii) SEQ ID NO: 25, 53, or 81, and (iv) SEQ ID NO: 28, 56, or 84; and (3) a V _L CDR3 having an amino acid sequence selected from the group consisting of ( _i ) SEQ ID NO: 20, 48, or 76, (ii) SEQ ID NO: 23, 51, or 79, and ( _iii ) SEQ ID NO: 26, 54, or 82.

In some embodiments, the antibody competes for binding to glycosylated BTLA with an antibody designated STC613, an antibody designated STC626, or an antibody designated STC635.

In some embodiments, the antibody specifically binds to a BTLA epitope comprising a sequence of five or more consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169.

In some embodiments, the antibody specifically binds to a BTLA epitope comprising one or more of the amino acids corresponding to R12, H16, K51, T57, S82, or S86 of BTLA of SEQ ID NO: 86.

In some embodiments, the antibody specifically binds to glycated BTLA with a dissociation constant (Kd) of less than 1 μM.

In some embodiments, the antibody specifically binds to glycosylated BTLA with a dissociation constant (Kd) of less than 100 nM, less than 10 nM, or less than 5 nM.

In some embodiments, the antibody specifically binds to glycosylated BTLA with a dissociation constant (Kd) of 5 nM or less.

In some embodiments, the antibody inhibits binding of herpesvirus-entry mediator (HVEM) to BTLA.

In some embodiments, the antibody inhibits HVEM binding with an IC50 of less than or equal to 1 μg/ml.

In some embodiments, the antibody inhibits HVEM binding with an IC50 of less than or equal to 0.8 μg/ml, less than or equal to 0.6 μg/ml, less than or equal to 0.4 μg/ml, or less than or equal to 0.2 μg/ml.

In some embodiments, the antibody inhibits HVEM binding with an IC50 of less than or equal to 0.2 μg/ml.

In some embodiments, the antibody is recombinant. In some embodiments, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In other embodiments, the antibody is Fab', F(ab')2, F(ab')3, a monovalent scFv, a bivalent scFv, a bispecific antibody, a bispecific scFv, or a single domain antibody. In some embodiments, the antibody is a human or humanized antibody. In further embodiments, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

In a further embodiment, the invention provides a composition comprising an antibody of the embodiment (e.g., an antibody that selectively binds to glycosylated BTLA over non-glycosylated BTLA) in a pharmaceutically acceptable carrier.

In another further embodiment, the invention provides an isolated polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous amino acids of human BTLA, wherein the fragment comprises at least one amino acid corresponding to position N75, N94, or N110 of human BTLA. In a further embodiment, the isolated polypeptide of the embodiment comprises a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous amino acids of human BTLA, comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated. In some embodiments, the polypeptide of the embodiment is fused or conjugated to an immunogenic polypeptide (e.g., keyhole limpet hemocyanin, KLH). In some embodiments, the polypeptide further comprises a Cys residue at the C- or N-terminus. For example, in some embodiments, the polypeptide is conjugated to the immunogenic polypeptide by a disulfide linkage at the Cys residue.

In yet another further embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated, and the polypeptide is formulated in a pharmaceutically acceptable carrier.

In another further embodiment, an immunogenic composition is provided comprising a polypeptide comprising a fragment of at least 7 consecutive amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated, and wherein the polypeptide is formulated in a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises an adjuvant, such as alum or Freund's adjuvant.

In yet another embodiment, the invention provides a method of treating a subject having cancer, comprising administering to the subject an effective amount of an antibody or isolated polypeptide of the embodiment. In some embodiments, the method of treating cancer comprises administering to the subject an effective amount of a polypeptide (e.g., a glycosylated BTLA polypeptide). In a further embodiment, the method of treating cancer comprises administering to the subject an effective amount of an antibody of the embodiment (e.g., the antibody selectively binds glycosylated BTLA over non-glycosylated BTLA). In some embodiments, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In some embodiments, the cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumor in adults, brain/CNS tumor in children, breast cancer, male breast cancer, cancer in adolescents, cancer in children, cancer in young adults, cancer of unknown primary, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophageal cancer, Ewing family tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal cancer or hypopharyngeal cancer, leukemia (e.g., adult acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), chronic myelomonocytic (CMML), childhood leukemia), liver cancer, lung cancer (e.g., non-small cell, small cell), Pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma in children, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., adult soft tissue cancer), skin cancer (e.g., stromal and squamous cell, melanoma, Merkel cell), small bowel cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms' tumor. In some embodiments, the antibody is in a pharmaceutically acceptable composition. In a further embodiment, the antibody is administered systemically. In specific embodiments, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

In some embodiments, the method further comprises administering to the subject at least a second anticancer treatment. In some embodiments, the second anticancer treatment is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

In yet another further embodiment, the invention provides a method of assessing BTLA glycosylation, N-linked glycosylation or N-glycosylation, comprising contacting a BTLA-containing sample with an antibody of the embodiment (e.g., the antibody selectively binds to glycosylated BTLA over non-glycosylated BTLA). In some embodiments, the method is an in vitro method . In some embodiments, the sample is a cell sample.

In yet another further embodiment, a method for producing an antibody is provided, comprising administering to an animal a polypeptide according to the embodiment (e.g., a polypeptide having a fragment of at least 7 consecutive amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated) and isolating the antibody from the animal. For example, the animal can be a mouse, a rat, a rabbit, or a human. In some embodiments, the method further comprises identifying CDRs of the antibody and humanizing sequences surrounding the CDRs to produce a humanized antibody. In yet another further embodiment, the method comprises recombinantly expressing the humanized antibody. Accordingly, in a further embodiment, the invention provides an isolated antibody produced by the method described above. Thus, in some embodiments, the invention provides an isolated antibody that selectively binds to a polypeptide of the invention (e.g., a fragment of at least 7 contiguous amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated) as compared to non-glycosylated BTLA.

6. Brief description of the drawing
The following drawings form a part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.
Figure 1a - Dot blot analysis of anti-BTLA mAbs; Experimental setup. Figure 1a is a schematic showing the setup of anti-BTLA mAb samples (STC601 - STC636) and controls (IgG control antibody; BTLA antibody commercially available from Biolegend, San Diego, CA) tested in the dot blot analysis. "PNGase F+" indicates endoglycosidase-treated BTLA; "PNGase F-" indicates untreated, glycated BTLA.
Figure 1b - Dot blot analysis of anti-BTLA mAbs; Experimental results. Figure 1b shows exemplary results of a dot blot analysis assessing sugar-specific BTLA binding of anti-BTLA mAbs STC601-STC636.
Figure 2 - Western blot analysis of anti-BTLA mAb. Figure 2 shows exemplary Western blot results showing binding of BTLA mAb to wild-type (WT) BTLA and BTLA mutants with a single N-glycosylation site (N75/2NQ, N94/2NQ, N116/2NQ) or no N-glycosylation site (3NQ).
Figure 3a - Surface plasmon resonance BTLA binding analysis; Figure 3a shows a sensorgram demonstrating BTLA titration experiment and BTLA binding to immobilized anti-BTLA mAb designated STC613.
Figure 3b - Surface plasmon resonance BTLA binding analysis; Figure 3b shows the sensorgrams showing BTLA titration experiments and BTLA binding to immobilized anti-BTLA mAb designated STC626.
Figure 3c - Surface plasmon resonance BTLA binding analysis; Figure 3c shows the sensorgrams showing BTLA titration experiments and BTLA binding to immobilized anti-BTLA mAb designated STC636.
Figure 4a - Binning of anti-BTLA mAb using STC613 . Figure 4a shows exemplary results of surface plasmon resonance experiments demonstrating binding of anti-BTLA mAb to BTLA-STC613 complexes.
Figure 4b - Binning of anti-BTLA mAb using STC636 . Figure 4b shows exemplary results of surface plasmon resonance experiments demonstrating binding of anti-BTLA mAb to BTLA-STC636 complexes.
Figure 5 - Neutralizing activity of anti-BTLA mAb. Figure 5 shows a bar diagram illustrating exemplary results of an ELISA assay analyzing BTLA:HVEM complex formation in the presence of 5 μg/ml or 0.5 μg/ml anti-BTLA mAb.
Figure 6 - Neutralizing activity of STC613 and STC626. Figure 6 is a graph showing titration curves for STC613 and STC626 in an ELISA-based BTLA:HVEM competition assay.
Figure 7 - BTLA epitope mapping of STC613. Figure 7 shows a graph showing the BTLA region and amino acid positions found to be cross-linked to STC613 in the BTLA-STC613 complex.

7. details

7.1. outline

BTLA was identified as an immunoglobulin domain-containing glycoprotein with two immunoreceptor tyrosine-based inhibitory motifs. BTLA is an inhibitory receptor on T lymphocytes that is not expressed by naive T cells but is induced during T cell activation. Watanabe et al. , Nature Immunology 4, 670-679(2003).

N-glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER) and subsequently processed in the Golgi apparatus (Schwarz & Aebi , Current Opinion in Structural Biology 21, 576-582(2011)). This type of modification is catalyzed by the membrane-associated oligosaccharyl transferase (OST) complex, which initially transfers preformed glycans composed of oligosaccharides to the asparagine (Asn) side chain acceptor located within the NXT motif (-Asn-X-Ser/Thr-) (Cheung and Reithmeier, Methods , 41(4): 451-59 (2007); Helenius and Aebi, Science 291(5512): 2364-69(2001)). Addition or removal of sugars from preformed glycans is mediated by groups of glycotransferases and glycosidases, respectively, which tightly regulate the N-glycosylation cascade in a cell- and location-dependent manner.

As used herein, and unless otherwise specified, the term "B- and T-lymphocyte attenuating factor" or "BTLA" refers to BTLA from any vertebrate source, including mammals such as primates (e.g., humans, cyno), dogs, and rodents (e.g., mice and rats). Unless otherwise specified, BTLA also includes various BTLA isoforms, related BTLA polypeptides, including SNP variants thereof, and other modified forms of BTLA, including but not limited to phosphorylated BTLA, glycosylated BTLA, and ubiquitinated BTLA.

An exemplary amino acid sequence of human BTLA is provided below, where the sites for N-linked glycosylation are underlined in bold (N75, N94, and N110):

As shown in the table below, all three N-glycosylation sites are located in the extracellular domain of BTLA.

The specific glycosylation site of a particular BTLA isoform or variant can vary from amino acids 75, 94, and 110 of that particular BTLA isoform or variant. In such circumstances, one of ordinary skill in the art would be able to determine the glycosylation site of any particular BTLA isoform or variant corresponding to N75, N94, and N110 of human BTLA as exemplified above based on the sequence alignment and other common sense in the art. Accordingly, the present invention also provides an antibody that selectively binds to a glycosylated form of a BTLA isoform or variant over an unglycosylated BTLA isoform or variant. The glycosylated site of the BTLA isoform or variant can be a site corresponding to N75, N94, and N110 of the human BTLA sequence provided above. The present invention also provides a polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous amino acids of a BTLA isoform or variant comprising at least one amino acid corresponding to position N75, N94, or N110 of the human BTLA sequence provided above.

As used herein, and unless otherwise specified, the articles "a," "an," and "the" refer to one or to more than one of the grammatical objects of the article. For example, an antibody refers to one antibody or to more than one antibody.

As used herein, and unless otherwise specified, the term "or" is used interchangeably with "and/or" unless explicitly indicated to refer only to alternatives or the alternatives are not mutually exclusive. As used herein, and unless otherwise specified, "another" refers to at least a second or more.

As used herein, and unless otherwise specified, the term "about" indicates that a value includes the inherent variation of error for the device or method used to determine that value, or the variation that exists between study subjects.

7.2. Antibodies and Polypeptides

As used herein, and unless otherwise specified, the term "antibody" refers to a polypeptide product of a B cell within the immunoglobulin (or "Ig") class of polypeptides capable of binding to a particular molecular antigen, such as IgG, IgM, IgA, IgD, IgE, and other molecules having an antigen-binding fragment. Antibodies may be composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), wherein the amino-terminal portion of each chain comprises a variable region of about 100 to about 130 amino acids or more and the carboxy-terminal portion of each chain comprises a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering , Second Edition, Oxford University Press.; Kuby (1997) Immunology , Third Edition, WH Freeman and Company, New York). Herein, the specific molecular antigen comprises glycosylated human BTLA. Antibodies provided in the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinantly produced antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, and anti-idiotypic (anti-Id) antibodies.

As used herein, and unless otherwise specified, the term "isolated" when used in reference to an antibody, antigen-binding fragment or polynucleotide means that the molecule referred to is free of at least one component with which it is found in nature. The term includes an antibody, antigen-binding fragment or polynucleotide that is free of some or all other components found in its natural environment. Components of the antibody's natural environment include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts and nutrients. Components of the antigen-binding fragment's or polynucleotide's natural environment include, for example, lipid membranes, organelles, proteins, nucleic acids, salts and nutrients. The antibodies, antigen-binding fragments or polynucleotides of the invention may also be free or substantially free of all or any of these components or other components of the cell from which they are isolated or recombinantly produced.

As used herein, and unless otherwise specified, the term "monoclonal antibody" refers to an antibody that is the product of a single cell clone or a hybridoma or a cell population derived from a single cell. Monoclonal antibody also refers to an antibody produced by recombinant methods from immunoglobulin genes encoding heavy and light chains to produce a single molecule immunoglobulin species. The amino acid sequences for the antibodies in a monoclonal antibody preparation are substantially homogeneous and the binding activity of the antibodies in such preparation exhibit substantially the same antigen binding activity. In contrast, polyclonal antibodies are obtained from different B cells within a population, and polyclonal antibodies are a combination of immunoglobulin molecules that bind to a specific antigen. Each immunoglobulin of a polyclonal antibody can bind to a different epitope of the same antigen. Methods for producing both monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide , W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

As used herein, and unless otherwise specified, the term "human antibody" refers to an antibody having a human variable region and/or a human constant region or a portion thereof corresponding to a human germline immunoglobulin sequence. Such human germline immunoglobulin sequences are disclosed by Kabat et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. Here, the human antibody may comprise an antibody encoded by a nucleic acid sequence that binds glycosylated human BTLA and is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence.

As used herein, and unless otherwise specified, the term "chimeric antibody" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al. , Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984)).

As used herein, and unless otherwise specified, the term "humanized antibody" refers to a chimeric antibody comprising a human immunoglobulin (e.g., an acceptor antibody) in which native complementarity determining region ("CDR") residues are replaced by residues from a corresponding CDR of a non-human species (e.g., a donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, humanized antibodies can have residues that are not found in the acceptor antibody or the donor antibody. These modifications are made to further improve antibody performance. A humanized antibody heavy or light chain can have substantially all of at least one variable region, wherein all or substantially all of the CDRs within the variable region correspond to CDRs of a non-human immunoglobulin and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. The humanized antibody can have at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. , Nature , 321:522-525 (1986); Riechmann et al. , Nature , 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol ., 2:593-596 (1992); Carter et al. , Proc. Natl. Acd. Sci. USA 89:4285-4289 (1992); and U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

As used herein, and unless otherwise specified, the term "recombinant antibody" refers to an antibody that is prepared, expressed, created, or isolated by recombinant means. A recombinant antibody can be an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, an antibody isolated from an animal (e.g., a mouse or a cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al. , Nucl. Acids Res . 20:6287-6295 (1992)), or an antibody prepared, expressed, created, or isolated by any other means involving splicing of immunoglobulin gene sequences to another DNA sequence. Such recombinant antibodies may have variable and constant regions, including those derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Humna Services, NIH Publication No. 91-3242). Recombinant antibodies may also be prepared by in vitro mutagenesis (or When animals that are transgenic for human Ig sequences are used, they can undergo in vivo somatic mutagenesis and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies , while derived from and related to human germline VH and VL sequences, may be sequences that do not naturally exist within the human antibody germline repertoire in vivo .

As used herein, and unless otherwise specified, a "neutralizing antibody" refers to an antibody that blocks the binding of BTLA to its natural ligand, e.g., herpesvirus-entry mediator (HVEM), and inhibits signaling pathways mediated by BTLA and/or other physiological activities thereof. The IC50 of a neutralizing antibody refers to the concentration of antibody required to neutralize 50% of BTLA in a neutralization assay, e.g., an ELISA assay that assays BTLA-HVEM complex formation. The IC50 of a neutralizing antibody can range from 0.01 to 10 μg/ml in a neutralization assay.

As used herein, and unless otherwise specified, the term "antigen-binding fragment" and similar terms refer to a portion of an antibody that comprises amino acid residues that immunospecifically bind to an antigen and confer to the antibody its specificity and affinity for the antigen. An antigen-binding fragment may be referred to as a functional fragment of an antibody. An antigen-binding fragment may be monovalent, bivalent, or multivalent.

Molecules having an antigen-binding fragment include, for example, Fd, Fv, Fab, F(ab'), F(ab) ₂ , F(ab') ₂ , F(ab) ₃ , F(ab') ₃ , single-chain Fv (scFv), diabody, triabody, tetrabody, minibody, or single-domain antibodies. The scFv can be a monovalent scFv or a bivalent scFv. Other molecules having an antigen-binding fragment can include, for example, a heavy chain or light chain polypeptide, a variable region polypeptide or a CDR polypeptide or a portion thereof, so long as such antigen-binding fragment retains binding activity. Such antigen-binding fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference , New York: VCH Publisher, Inc.; Huston et al. , Cell Biophysics , 22:189-224 (1993); pl ckthun and Skerra, Meth. Enzymol ., 178:497-515 (1989) and Day, ED, Advanced Immunochemistry , Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antigen-binding fragment can be a polypeptide having an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

The heavy chain of an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion comprises a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion comprises a constant region. The constant region can be one of five distinct types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ, and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these distinct types of heavy chains produce the five well-known classes of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The heavy chains can be human heavy chains.

The light chain of an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion comprises a variable region of about 100 to about 110 or more amino acids and the carboxy-terminal portion comprises a constant region. The approximate length of the light chain is 211 to 217 amino acids. There are two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

The variable domain or variable region of an antibody is a portion of the light or heavy chain of an antibody, typically located at the amino-terminus of the light or heavy chain and typically about 120 to 130 amino acids in the heavy chain or about 100 to 110 amino acids in the light chain, that is used in the binding and specificity of each particular antibody for its particular antigen. The variable domains vary widely in sequence between different antibodies. The variability in sequence is concentrated in the CDRs, while the less variable portions of the variable domain are called the framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. The numbering of amino acid positions as used herein follows the EU index, as in Kabat et al. (1991) Sequences of proteins of immunological interest . (US Department of Health and Human Services, Washington, DC) 5 ^th ed. The variable region may be a human variable region.

CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and were defined by Kabat, for example, as the most hypervariable regions within an antibody variable (V) domain (Kabat et al. , J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). The CDR region sequences were also structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and can therefore assume different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terms are well recognized in the art. The positions of the CDRs within the canonical antibody variable domains have been determined by comparison of many structures (Al-Lazikani et al. , J. Mol. Biol. 273:927-948 (1997); Morea et al. , Methods 20:267-279 (2000)). Since the number of residues within the hypervariable region varies in different antibodies, additional residues at canonical positions are conventionally numbered a, b, c, etc., adjacent to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al. , supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to standard nomenclature are disclosed in Table 1 below.

Table 1: CDR Definitions

One or more CDRs may also be covalently or non-covalently incorporated into the molecule, thereby rendering the molecule an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) non-covalently. The CDRs enable the immunoadhesin to bind to a specific antigen of interest.

"Framework" or "FR" residues refer to variable domain residues flanking a CDR. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues as defined herein.

As used herein, and unless otherwise specified, the term "isolated" as used with respect to an antibody means that the antibody is substantially free of cellular material or other contaminating proteins and/or other contaminating components from the cell or tissue source from which the antibody is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The language "substantially free of cellular material" includes antibody preparations in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody substantially free of cellular material includes an antibody preparation having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). In some embodiments, when the antibody is recombinantly produced, the antibody is substantially free of culture medium, e.g., the culture medium comprises less than about 20%, 10%, or 5% of the volume of the protein preparation. In some embodiments, where the antibody is produced by chemical synthesis, the antibody is substantially free of chemical precursors or other chemicals, for example, the antibody is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such antibody preparations contain less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminants may also include, but are not limited to, substances that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified to (1) greater than 95% antibody by weight as determined by the Lowry method (Lowry et al . J. Bio. Chem. 193: 265-275, 1951), such as 99%, (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes an antibody in situ within a recombinant cell, since at least one component of the antibody's native environment will not be present. Generally, however, an isolated antibody will be prepared by at least one purification step. In specific embodiments, the antibodies provided herein are isolated.

As used herein, and unless otherwise specified, the terms “polynucleotide,” “nucleotide,” “nucleic acid,” “nucleic acid molecule,” and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

As used herein, and unless otherwise specified, the term "isolated" when used in reference to a nucleic acid molecule means that the nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, the nucleic acid molecule(s) encoding an antibody provided herein are isolated or purified.

As used herein and unless otherwise specified, the terms "bind" or "binding" refer to an interaction between molecules. The interaction can be a noncovalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interaction, and/or van der Waals interaction. The strength of the overall noncovalent interaction between an antibody and a single epitope of a target molecule, such as glycosylated human BTLA, is the affinity of the antibody for that epitope. "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen).

The affinity of a binding molecule X, such as an antibody, for its binding partner Y, e.g., its conjugate antigen, can generally be represented by the dissociation constant (K _D ). Low affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high affinity antibodies generally bind antigen more rapidly and tend to remain bound longer. A variety of methods are known in the art for measuring binding affinity, any of which can be used for the purposes of the present invention. The "K _D " or "K _D value" can be measured by assays known in the art, e.g., by binding assays. K _D can be measured, for example, in a radiolabeled antigen binding assay (RIA) performed with a Fab version of an antibody of interest and its antigen (Chen, et al. , (1999) J. Mol. Biol. 293:865-881). K _D or K _D values can also be measured by surface plasmon resonance analysis by Biacore, for example, using a BIAcoreTM-2000 or BiacoreTM-3000 (BIAcore, Inc., Piscataway, NJ), or by biolayer interferometry, for example, using an OctetQK384 system (ForteBio, Menlo Park, Calif.). As used herein, and unless otherwise specified, an antibody is said to "selectively bind" a first molecular antigen over a second molecular antigen if the antibody binds to the first molecular antigen with higher affinity than to the second molecular antigen. Antibodies will not generally bind to completely unrelated antigens.

As used herein, and unless otherwise specified, the term "polypeptide" as used herein includes oligopeptides having from 2 to 30 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25 or 30 amino acids) and longer amino acid chains, e.g., greater than 30 amino acids, greater than 50 amino acids, greater than 100 amino acids, greater than 150 amino acids, greater than 200 amino acids, greater than 300 amino acids, greater than 400 amino acids, greater than 500 amino acids, or greater than 600 amino acids. Polypeptides can be produced, for example, by recombinant expression or chemical synthesis. Polypeptides of the invention can be modified post-translationally or chemically (e.g., by glycosylation, carbamylation, phosphorylation, biotinylation, attachment of a fluorescent dye, etc.). Polypeptides can be glycosylated at specific sites. Polypeptides can include non-natural amino acids that are not encoded by the natural genetic code. For example, polypeptides can include methylated backbone structures, peptoid backbone structures (poly-N-substituted glycines), L-amino acids, R-amino acids, etc. Polypeptides can include wild-type sequences, naturally occurring variant sequences, mutant sequences (e.g., point mutants, deletion mutants), etc.

As used herein, and unless otherwise specified, the term "vector" refers to a material used to introduce a nucleic acid molecule into a host cell. Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, wherein the artificial chromosome may include a selection sequence or marker operable for stable integration into the chromosome of the host cell. Additionally, the vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, supplement nutrient deficiencies, or supply important nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and others well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., both antibody heavy and light chains), both nucleic acids may be inserted, for example, into one expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acid may be operably linked to a common expression control sequence or may be linked to different expression control sequences, such as an inducible promoter and a constitutive promoter. Introduction of the nucleic acid molecule into the host cell can be confirmed using methods well known in the art. Such methods include nucleic acid analysis, such as, for example, Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of a gene product, or other suitable analytical methods for testing for expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will appreciate that a nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., an anti-BTLA antibody provided herein), and it will further be appreciated that the level of expression can be optimized to obtain sufficient expression using methods well known in the art.

As used herein, and unless otherwise specified, the term "host cell" refers to a specific subject cell that has been transfected with a nucleic acid molecule and the descendants or potential descendants of such a cell. The descendants of such a cell may not be identical to the parent cell that was transfected with the nucleic acid molecule, due to mutations that may occur in subsequent generations, environmental influences, or integration of the nucleic acid molecule into the host cell genome.

7.2.1. Anti-glycated BTLA antibodies

The present invention provides an isolated antibody that selectively binds to glycosylated BTLA over non-glycosylated BTLA. The BTLA can be human BTLA. The glycosylated BTLA can be a specific N-glycan structure of BTLA or a glycopeptide of BTLA. In some embodiments, the antibody provided herein is an antigen-binding fragment that selectively binds to glycosylated BTLA over non-glycosylated BTLA.

In some embodiments, the isolated antibody provided herein selectively binds human BTLA that is glycosylated at N75, N94, N110 or any combination thereof, as compared to non-glycosylated BTLA. In some embodiments, the isolated antibody selectively binds human BTLA having N75 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N94 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N110 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N75 and N94 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N94 and N110 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N75 and N110 glycosylation. In some embodiments, the isolated antibody selectively binds human BTLA having N75, N94 and N110 glycosylation.

In some embodiments, the isolated antibody provided herein specifically masks a glycosylation motif of BTLA comprising BTLA positions N75, N94, N110, or any combination thereof. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising position N75 of human BTLA. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising position N94 of human BTLA. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising position N110 of human BTLA. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising positions N75 and N94 of human BTLA. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising positions N75 and N110 of human BTLA. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising positions N94 and N110 of human BTLA. In some embodiments, the isolated antibody specifically masks a glycosylation motif of BTLA comprising positions N75, N94, and N110 of human BTLA.

In some embodiments, the antibody provided herein selectively binds to one or more glycosylation motifs of BTLA. In some embodiments, the antibody selectively binds to a glycopeptide having a glycosylation motif and an adjacent peptide. In some embodiments, the antibody selectively binds to glycosylated BTLA with a K _D that is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the K _D shown for non-glycosylated BTLA. In some embodiments, the antigen-binding fragment binds to glycosylated BTLA with a K _D that is less than 50% of the K _D shown for non-glycosylated BTLA. In some embodiments, the antibody binds to glycosylated BTLA with a _Kd that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50% the Kd shown for nonglycosylated BTLA. In a further aspect, the antibody binds to glycosylated BTLA with a _Kd that is at least 10-fold less than the _Kd shown for nonglycosylated BTLA.

In some embodiments, selective binding of an antibody provided herein to glycosylated BTLA over non-glycosylated BTLA is determined using, for example, measurements of fluorescence intensity (e.g., MFI) in a FACS analysis or an ELISA. See, for example, Examples 1 and 3. In some embodiments, the measured fluorescence intensity (e.g., MFI) is indicative of binding of a fluorescently labeled antibody provided herein (e.g., a FITC labeled antibody) to glycosylated or non-glycosylated BTLA (e.g., cell surface expressed BTLA, BTLA immobilized on a surface or bead, or BTLA in bulk solution). In some embodiments, binding of the antibody to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 120-fold, at least 140-fold, at least 160-fold, at least 180-fold, or at least 200-fold greater than the fluorescence intensity exhibited by non-glycosylated BTLA. See, e.g., Table 10. In some embodiments, binding of the antibody to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than the fluorescence intensity exhibited by non-glycosylated BTLA. In some embodiments, binding of the antibody to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, or at least 20-fold greater than the fluorescence intensity exhibited by non-glycosylated BTLA. In some embodiments, binding of the antibody to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 10 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times greater than the fluorescence intensity exhibited by non-glycosylated BTLA. In some embodiments, binding of the antibody to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 100 times, at least 120 times, at least 140 times, at least 160 times, at least 180 times, or at least 200 times greater than the fluorescence intensity exhibited by non-glycosylated BTLA.

In some embodiments, the anti-glycosylated BTLA antibody provided herein can be IgG, IgM, IgA, IgD, or IgE. The anti-glycosylated BTLA antibody can also be a chimeric antibody, an affinity matured antibody, a humanized antibody, or a human antibody. The anti-glycosylated BTLA antibody can also be a camelized antibody, an intrabody, an anti-idiotypic (anti-Id) antibody. In some embodiments, the anti-glycosylated BTLA antibody can be a polyclonal antibody or a monoclonal antibody.

In some embodiments, the antibody provided in the present invention is an antigen-binding fragment that selectively binds to glycosylated BTLA over non-glycosylated BTLA. The antigen-binding fragment is selected from the group consisting of Fd, Fv, Fab, F(ab'), F(ab) ₂ , F(ab') ₂ , It can be F(ab) ₃ , F(ab') ₃ , single-chain Fv (scFv), diabody, triabody, tetrabody, minibody or single-domain antibody. The scFv can be monovalent scFv or bivalent scFv.

Several exemplary mouse monoclonal antibodies (mAbs) that selectively bind to glycosylated BTLA over nonglycosylated BTLA have been produced and characterized. See, e.g., Examples 1-7. Exemplary anti-BTLA mAbs include IgG1, IgG2A, IgG2B, IgG3, and IgGM isotypes. See, e.g., Table 8. For example, antibodies designated STC604, STC605, STC606, STC608, STC610, STC613, STC618, STC622, STC626, STC627, STC628, STC630, and STC636 demonstrate glycosylation-specific binding to BTLA. See, e.g., FIGS. 1 and 2 . For example, antibodies designated STC604, STC610, STC613, STC618, STC622, STC626, and STC635 bind BTLA with high affinity, with KDs ranging from 0.256 nM (STC613) to 5.61 nM (STC635). See, e.g., Table 11. For example, antibodies designated STC613 and STC626 inhibit BTLA binding to its natural ligand HVEM with IC50s of 1.088 μg/ml (STC613) and 0.416 μg/ml (STC626). See, e.g., FIG. 6. The BTLA epitope of one exemplary anti-BTLA mAb designated STC613 is also provided herein. Accordingly, the present invention provides neutralizing anti-BTLA mAbs having specific sequence characteristics, anti-BTLA mAbs that sugar-specifically bind to BTLA, and specific BTLA epitopes and their uses in cancer therapy.

In some embodiments, the anti-glycated BTLA antibody provided herein comprises a VH region, a VL region, a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and/or a VL CDR3 of any one of the monoclonal antibodies disclosed herein (e.g., STC613, STC626, or STC635), wherein the amino acid sequences are set forth in Tables 2-7. Accordingly, in some embodiments, the antibody provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from (a) the antibody designated STC613; (b) the antibody designated STC626, or (c) the antibody designated STC635, as shown in Tables 3, 5, and 7.

The antibody designated STC613 comprises a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 3.

The antibody designated STC626 comprises a VH sequence of SEQ ID NO: 30 and a VL sequence of SEQ ID NO: 32.

The antibody designated STC635 comprises a VH sequence of SEQ ID NO: 58 and a VL sequence of SEQ ID NO: 60.

Table 2: Sequences of the heavy chain variable (VH) and light chain variable (VL) regions of the mouse monoclonal anti-human BTLA antibody STC613.

Table 3: CDR sequences of mouse monoclonal anti-human BTLA antibody STC613

Table 4: Sequences of the heavy chain variable (VH) and light chain variable (VL) regions of the mouse monoclonal anti-human BTLA antibody STC626

Table 5: CDR sequences of mouse monoclonal anti-human BTLA antibody STC626

Table 6: Sequences of the heavy chain variable (VH) and light chain variable (VL) regions of the mouse monoclonal anti-human BTLA antibody STC635

Table 7: CDR sequences of mouse monoclonal anti-human BTLA antibody STC635

In some embodiments, the anti-glycated BTLA antibody provided herein comprises a VH region or a VH domain. In other embodiments, the antibody provided herein comprises a VL region or a VL chain. In some embodiments, the antibody provided herein has (i) a VH domain or a VH region; and/or (ii) a combination of a VL domain or a VL region.

In some embodiments, the anti-glycated BTLA antibody provided herein comprises or consists of six CDRs, e.g., a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and/or a VL CDR3 identified in Table 3, 5, or 7. In some embodiments, the antibody provided herein can comprise fewer than six CDRs. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and/or a VL CDR3 identified in Table 3, 5, or 7. In some embodiments, the antibody is an antibody designated as STC613 as disclosed herein; (b) an antibody designated as STC626; Or (c) comprises or consists of 1, 2, 3, 4, or 5 CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a murine monoclonal antibody selected from the group consisting of antibodies designated STC635. Thus, in some embodiments, the antibody comprises or consists of 1, 2, 3, 4, or 5 CDRs of any one of a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and/or a VL CDR3 identified in Tables 3, 5, or 7.

In some embodiments, an antibody provided herein comprises one or more (e.g., one, two, or three) VH CDRs listed in Table 3, 5, or 7. In other embodiments, an antibody provided herein comprises one or more (e.g., one, two, or three) VL CDRs listed in Table 3, 5, or 7. In yet other embodiments, an antibody provided herein comprises one or more (e.g., one, two, or three) VH CDRs listed in Table 3, 5, or 7 and one or more VL CDRs listed in Table 3, 5, or 7. Accordingly, in some embodiments, the antibody comprises a VH CDR1 having an amino acid sequence of any one of SEQ ID NOs: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71. In another embodiment, the antibody comprises a VH CDR2 having an amino acid sequence of any one of SEQ ID NOs: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72. In other embodiments, the antibody comprises a VH CDR3 having an amino acid sequence of any one of SEQ ID NOs: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73. In some embodiments, the antibody comprises a VH CDR1 and/or VH CDR2 and/or VH CDR3 independently selected from a VH CDR1, a VH CDR2, a VH CDR3 set forth in any one of the amino acid sequences set forth in Tables 3, 5, or 7. In some embodiments, the antibody comprises a VL CDR1 having an amino acid sequence of any one of SEQ ID NOs: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83. In other embodiments, the antibody comprises a VL CDR2 having an amino acid sequence of any one of SEQ ID NOs: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84. In other embodiments, the antibody comprises a VL CDR3 having an amino acid sequence of any one of SEQ ID NOs: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85. In some embodiments, the antibody comprises a VL CDR1 and/or VL CDR2 and/or VL CDR3 independently selected from a VL CDR1, a VL CDR2, a VL CDR3 set forth in any one of the amino acid sequences set forth in Tables 3, 5, or 7.

In some embodiments, the antibody provided herein comprises (1) a VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 6, 34, or 62, (ii) SEQ ID NO: 9, 37, or 65, (iii) SEQ ID NO: 12, 40, or 68, and (iv) SEQ ID NO: 15, 43, or 71; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 7, 35, or 63, (ii) SEQ ID NO: 10, 38, or 66, (iii) SEQ ID NO: 13, 41, or 69, and (iv) SEQ ID NO: 16, 44, or 72; and (3) (i) SEQ ID NO: 8, 36, or 64; (ii) SEQ ID NO: 11, 39, or 67; (iii) a heavy chain variable (VH) region comprising a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 42, or 70, and (iv) SEQ ID NO: 17, 45, or 73; and/or (1) a VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 18, 46, or 74; (ii) SEQ ID NO: 21, 49, or 77; (iii) SEQ ID NO: 24, 52, or 80, and (iv) SEQ ID NO: 27, 55, or 83; (2) a light chain variable (VL) region comprising a VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 19, 47, or 75, (ii) SEQ ID NO: 22, 50, or 78, (iii) SEQ ID NO: 25, 53, or 81, and (iv) SEQ ID NO: 28, 56, or 84; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 20, 48, or 76, (ii) SEQ ID NO: 23, 51, or 79, (iii) SEQ ID NO: 26, 54, or 82, and (iv) SEQ ID NO: 29, 57, or 85.

In some embodiments, the antibody provided herein comprises (1) a VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 6, 34, or 62, (ii) SEQ ID NO: 9, 37, or 65, (iii) SEQ ID NO: 12, 40, or 68, and (iv) SEQ ID NO: 15, 43, or 71; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 7, 35, or 63, (ii) SEQ ID NO: 10, 38, or 66, (iii) SEQ ID NO: 13, 41, or 69, and (iv) SEQ ID NO: 16, 44, or 72; and (3) (i) SEQ ID NO: 8, 36, or 64; (ii) SEQ ID NO: 11, 39, or 67; (iii) a heavy chain variable (VH) region comprising a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 42, or 70, and (iv) SEQ ID NO: 17, 45, or 73.

In some embodiments, the antibody provided herein comprises (1) a VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 18, 46, or 74; (ii) SEQ ID NO: 21, 49, or 77; (iii) SEQ ID NO: 24, 52, or 80, and (iv) SEQ ID NO: 27, 55, or 83; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 19, 47, or 75, (ii) SEQ ID NO: 22, 50, or 78, (iii) SEQ ID NO: 25, 53, or 81, and (iv) SEQ ID NO: 28, 56, or 84; and (3) a light chain variable (VL) region comprising a VL CDR3: having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 20, 48, or 76, (ii) SEQ ID NO: 23, 51, or 79, (iii) SEQ ID NO: 26, 54, or 82, and (iv) SEQ ID NO: 29, 57, or 85.

The invention also provides an antibody comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 3, 5, or 7. Specifically, the invention provides an antibody comprising a VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71) and a VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83) listed in Tables 3, 5, and 7; VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71) and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71) and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72) and VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72) and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72) and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73) and VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83); VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73) and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73) and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), and VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), and VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73) and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), and VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83) and VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83) and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84) and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR1 (SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); VH CDR2 (SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72), VH CDR3 (SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73), VL CDR1 (SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83), VL CDR2 (SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84), and VL CDR3 (SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85); or a VH CDR (SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73) and a VL CDR (SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 46, 47, 48, 49, 50, 51, 52, 53, 54, Provided is an antibody comprising any combination of: 55, 56, 57, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73. In some embodiments, the heavy chain variable (VH) region comprises (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71; and (2) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72. In some embodiments, the heavy chain variable (VH) region comprises (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73. In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73.

In some embodiments, the antibody provided herein comprises a heavy chain variable (VH) region having a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 6, 9, 12, 15, 34, 37, 40, 43, 62, 65, 68, or 71. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 6. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 9. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 12. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 15. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 34. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 37. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 40. The VH CDR1 can comprise the amino acid sequence of SEQ ID NO: 43. The VH CDR1 may comprise the amino acid sequence of SEQ ID NO: 62. The VH CDR1 may comprise the amino acid sequence of SEQ ID NO: 65. The VH CDR1 may comprise the amino acid sequence of SEQ ID NO: 68. The VH CDR1 may comprise the amino acid sequence of SEQ ID NO: 71.

In some embodiments, the antibody provided herein comprises a heavy chain variable (VH) region having a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, 16, 35, 38, 41, 44, 63, 66, 69, or 72. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 7. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 10. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 13. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 16. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 35. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 38. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 41. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 44. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 63. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 66. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 69. The VH CDR2 can comprise the amino acid sequence of SEQ ID NO: 72.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region having a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 36, 39, 42, 45, 64, 67, 70, or 73. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 8. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 11. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 14. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 17. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 36. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 39. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 42. The VH CDR3 can comprise the amino acid sequence of SEQ ID NO: 45. The VH CDR3 may comprise the amino acid sequence of SEQ ID NO: 64. The VH CDR3 may comprise the amino acid sequence of SEQ ID NO: 67. The VH CDR3 may comprise the amino acid sequence of SEQ ID NO: 70. The VH CDR3 may comprise the amino acid sequence of SEQ ID NO: 73.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the antibody provided herein has an antigen-binding fragment having a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 34; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 35; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 64.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 65; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 67.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 68; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 71; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 2. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 30. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 58. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, an antibody provided herein has a light chain variable (VL) region comprising (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83; and (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84. In some embodiments, an antibody provided herein has a light chain variable (VL) region comprising (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83; and (3) a light chain variable (VL) region comprising a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85. In some embodiments, the antibody provided herein has a light chain variable (VL) region comprising (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, 21, 24, 27, 46, 49, 52, 55, 74, 77, 80, or 83. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 18. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 21. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 24. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 27. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 46. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 49. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 52. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 55. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 74. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 77. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 80. The VL CDR1 can comprise the amino acid sequence of SEQ ID NO: 83.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, 22, 25, 28, 47, 50, 53, 56, 75, 78, 81, or 84. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 19. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 22. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 25. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 28. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 47. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 50. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 53. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 56. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 75. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 78. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 81. The VL CDR2 can comprise the amino acid sequence of SEQ ID NO: 84.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20, 23, 26, 29, 48, 51, 54, 57, 76, 79, 82, or 85. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 20. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 23. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 26. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 29. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 48. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 51. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 54. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 57. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 76. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 79. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 82. The VL CDR3 can comprise the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 27; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 28; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 46; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 47; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 48.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 49; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 50; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 51.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 55; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 74; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 75; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 76.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 77; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 78; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 79.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 80; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 82.

In some embodiments, the antibody provided herein has a light chain variable (VL) region having (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 83; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 84; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the antibody provided herein has a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 4. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, the antibody provided herein has a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, the antibody provided herein has a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 60. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and/or (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and/or (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 26. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 27; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 28; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 29. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 46; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 47; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 48. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 49; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 50; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 51. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 55; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 64; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 74; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 75; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 76. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 65; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 67; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 77; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 78; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 79. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 68; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 80; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 82. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a heavy chain variable (VH) region comprising: (a) (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 71; (2) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and (3) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 73; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 83; (2) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 84; and (3) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 85. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a VH region comprising the amino acid sequence of SEQ ID NO: 2 and a VL region having the amino acid sequence of SEQ ID NO: 4. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, the antibody provided herein has a VH region having the amino acid sequence of SEQ ID NO: 30 and a VL region having the amino acid sequence of SEQ ID NO: 32. The molecule can be an antibody. The antibody can be a monoclonal antibody. The antibody can be a humanized antibody.

In some embodiments, the antibody provided herein has a VH region having an amino acid sequence of SEQ ID NO: 58 and a VL region having an amino acid sequence of SEQ ID NO: 60. The antibody may be a monoclonal antibody. The antibodies may be monoclonal antibodies. The antibodies may be humanized antibodies.

In some embodiments, the antibody provided herein is a mouse monoclonal antibody designated STC613, or a humanized antibody version thereof. The humanized STC613 antibody can comprise the VH region, the VL region, or both the VH and VL regions of STC613 disclosed herein. The humanized STC613 antibody can also comprise the six CDR regions of STC613 disclosed herein (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3). The humanized STC613 antibody can also comprise less than six CDR regions of STC613. In some embodiments, the humanized STC613 antibody may also comprise one, two, three, four, or five CDR regions of STC613 (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3).

In some embodiments, the antibody provided herein is a mouse monoclonal antibody designated STC626, or a humanized antibody version thereof. The humanized STC626 antibody can comprise the VH region, the VL region, or both the VH and VL regions of STC626 disclosed herein. The humanized STC626 antibody can also comprise the six CDR regions of STC626 disclosed herein (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3). The humanized STC626 antibody can also comprise less than six CDR regions of STC626. In some embodiments, the humanized STC626 antibody may also comprise one, two, three, four, or five CDR regions of STC626 (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3).

In some embodiments, the antibody provided herein is a mouse monoclonal antibody designated STC635, or a humanized antibody version thereof. The humanized STC635 antibody can comprise the VH region, the VL region, or both the VH and VL regions of STC635 disclosed herein. The humanized STC635 antibody can also comprise the six CDR regions of STC635 disclosed herein (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3). The humanized STC635 antibody can also comprise less than six CDR regions of STC635. In some embodiments, the humanized STC635 antibody may also comprise one, two, three, four, or five CDR regions of STC635 (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3).

In some embodiments, the anti-BTLA antibody provided herein is IgG, IgM, IgA, IgD, or IgE. In some embodiments, the anti-BTLA antibody provided herein is IgG1, IgG2A, IgG2B, IgG3, or IgGM.

Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequence encoding the antigen-binding fragment, or antibody, provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. In some embodiments, the derivative comprises fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In specific embodiments, the derivative has a conservative amino acid substitution made at one or more predicted nonessential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a side chain having a similar charge. Families of amino acid residues having side chains having similar charges are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and its activity can be determined.

In some embodiments, the anti-BTLA antibodies provided herein specifically bind to BTLA and have an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of murine monoclonal antibody STC613, STC626, or STC635, or an antigen-binding fragment thereof, such as a VH domain or a VL domain. In some embodiments, the anti-BTLA antibodies provided herein can have an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence set forth in SEQ ID NO: 2, 4, 30, 32, 58, or 60. In some embodiments, the anti-BTLA antibodies provided herein can have VH CDR and/or VL CDR amino acid sequences that are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a VH CDR amino acid sequence and/or a VL CDR amino acid sequence disclosed in Tables 3, 5, or 7.

In some embodiments, the anti-BTLA antibodies provided herein are hybridized under stringent conditions (e.g. , hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under very stringent conditions (e.g., hybridization to filter-bound nucleic acids in 6X SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions known to those of skill in the art (see, e.g., Ausubel, FM et al. , eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). NOTE) It may have an amino acid sequence of a VH domain and/or an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH and/or VL domains disclosed in Table 2, 4 or 6.

In some embodiments, the anti-BTLA antibodies provided herein are hybridized under stringent conditions (e.g. , hybridization to filter-bound DNA in 6XSSC at about 45°C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65°C), under very stringent conditions (e.g., hybridization to filter-bound nucleic acids in 6XSSC at about 45°C followed by one or more washes in 0.1xSSC/0.2% SDS at about 68°C), or under other stringent hybridization conditions known to those of skill in the art (see, e.g., Ausubel, FM et al. , eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3) as set forth in Table 2, 4 or 6. It may have an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding either one of the disclosed VH CDRs and/or VL CDRs.

In some embodiments, the invention also provides an isolated nucleic acid that hybridizes to the complement of a nucleotide sequence encoding any one of the VH CDRs and/or VL CDRs disclosed in Tables 2, 4 or 6, or under stringent conditions (e.g. , hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6X SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions known to those of skill in the art.

In some embodiments, the invention also provides an isolated nucleic acid that hybridizes to the complement of a nucleotide sequence encoding any one of the VH and/or VL domains disclosed in Tables 2, 4, or 6, under stringent conditions (e.g. , hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2x SSC/0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6X SSC at about 45° C. followed by one or more washes in 0.1x SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions known to those of skill in the art.

In some embodiments, the isolated nucleic acid can have the sequence of SEQ ID NO: 3, 31 or 59, or a sequence that hybridizes to the complement of the nucleotide sequence of SEQ ID NO: 3, 31 or 59 under stringent conditions (e.g. , hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6X SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions known to those of skill in the art.

In some embodiments, the isolated nucleic acid has the sequence of SEQ ID NO: 5, 33 or 61, or can have a sequence that hybridizes to the complement of the nucleotide sequence of SEQ ID NO: 5, 33 or 61 under stringent conditions (e.g. , hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under very stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6X SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions known to those of skill in the art.

In some embodiments, the anti-glycated BTLA antibodies provided herein can be chemically modified, for example, by covalent attachment of any type of molecule to the antibody. For example, but without limitation, antibody derivatives include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, and the like. Any of the many chemical modifications can be performed by known techniques, including but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody can contain one or more unconventional amino acids.

The anti-glycated BTLA antibodies provided herein can have a framework region known to those of skill in the art (e.g., human or non-human fragments). The framework region can be, for example, a naturally occurring or consensus framework region. In a specific embodiment, the framework region of the antibodies provided herein is human (see, e.g., Chothia et al. , 1998, J. Mol. Biol. 278:457-479, which is incorporated herein by reference in its entirety, for a list of human framework regions). See also Kabat et al. (1991) Sequences of Proteins of Immunological Interest (US Department of Health and Human Services, Washington, DC) 5th ed.

The BTLA epitope of STC613 was mapped by crosslinking analysis. See Example 7. Table 8 summarizes the crosslinked peptides of BTLA and STC613, which represent the BTLA epitope of STC613 (SEQ ID NOs: 161, 162, 163, 164, 165, 166). Figure 6 shows the synthetic epitope of the BTLA antigen (SEQ ID NO: 86) for STC613:

IKRQSEHSILA (SEQ ID NO: 167) ― VKLEDRQTSWK (SEQ ID NO: 168) - NGSYRCSANFQ (SEQ ID NO: 169)

Table 8: Cross-linked peptides of BTLA (SEQ ID NO: 86) with STC613 analyzed by nLC-orbitrap MS/MS.

* Peptide sequence positions are indicated for the STC613 amino acid sequence (protein 1) of SEQ ID NO: 2 and 4, and the BTLA amino acid sequence (protein 2) of SEQ ID NO: 86.

Accordingly, the present invention also provides anti-glycated BTLA antibodies that competitively block (e.g., in a dose-dependent manner) a BTLA epitope disclosed herein. In some embodiments, the present invention provides anti-glycated BTLA antibodies that competitively block (e.g., in a dose-dependent manner) a BTLA epitope of STC613 disclosed herein. In some embodiments, an anti-glycated BTLA antibody provided herein specifically binds to an epitope of BTLA disclosed herein. In some embodiments, an anti-glycated BTLA antibody provided herein specifically binds to a BTLA epitope of STC613.

In some embodiments, the anti-glycated BTLA antibody provided herein competitively blocks (e.g., in a dose-dependent manner) a BTLA epitope, wherein the BTLA epitope has at least 5 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The BTLA epitope can have at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 6 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 7 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 8 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 9 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 10 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 11 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 12 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 13 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 14 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have at least 15 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The anti-glycated BTLA antibody can be a humanized antibody.

In some embodiments, the anti-glycated BTLA antibody competitively blocks (e.g., in a dose-dependent manner) a BTLA epitope, wherein the BTLA epitope has the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. In some embodiments, the anti-glycated BTLA antibody provided herein has an antigen binding fragment that specifically binds to an epitope of BTLA, wherein the BTLA epitope has the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 161. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 162. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 163. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 164. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 165. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 166. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 167. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 168. The epitope of BTLA can have the amino acid sequence of SEQ ID NO: 169.

In some embodiments, the anti-glycated BTLA antibody provided herein competitively blocks (e.g., in a dose-dependent manner) a BTLA epitope, wherein the BTLA epitope has one or more amino acids of R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The BTLA epitope can have one, two, three, four, or five amino acids of R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The BTLA epitope can have one amino acid of R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have two amino acids among R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have three amino acids among R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have four amino acids among R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have five amino acids among R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The anti-glycosylated BTLA antibody can be a humanized antibody.

In some embodiments, the anti-glycated BTLA antibody provided herein competitively blocks (e.g., in a dose-dependent manner) a BTLA epitope, wherein the BTLA epitope has one or more amino acids of R12, H16, K51, T57, S82, or S86 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have R12 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have H16 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have K51 of BTLA (SEQ ID NO: 86). The epitope of BTLA can have T57 of BTLA (SEQ ID NO: 86). The epitope of BTLA may have S82 of BTLA (SEQ ID NO: 86). The epitope of BTLA may have S86 of BTLA (SEQ ID NO: 86). The anti-glycated BTLA antibody may be a humanized antibody.

In some embodiments, the anti-glycated BTLA antibodies provided herein have high affinity for glycosylated BTLA or a polypeptide, or polypeptide fragment or epitope thereof. In one embodiment, the molecules provided herein can be anti-BTLA antibodies having higher affinity for BTLA than known antibodies (e.g., commercially available monoclonal antibodies discussed elsewhere herein). In some embodiments, the anti-BTLA antibodies provided herein can have an affinity for the BTLA antigen that is 2- to 10-fold (or more) higher than known anti-BTLA antibodies, as assessed by techniques disclosed herein or known to one of skill in the art (e.g., Biacore analysis). According to these embodiments, in one embodiment, the affinity of the antibodies is assessed by Biacore analysis.

In some embodiments, the anti-glycosylated BTLA antibodies provided herein can specifically bind to glycated BTLA or a glycosylated polypeptide fragment or an epitope thereof with a dissociation constant (K _D ) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In some embodiments, the anti-glycosylated BTLA antibodies provided herein have a K _D of 500 nM or less. In some embodiments, the anti-glycosylated BTLA antibodies provided herein have a K _D of 200 nM or less. In some embodiments, the anti-glycosylated BTLA antibodies provided herein have a K _D of 100 nM or less. In some embodiments, the anti-glycosylated BTLA antibodies provided herein have a K _D of 50 nM or less. In some embodiments, the anti-glycosylated BTLA antibodies provided herein have a K _D of 20 nM or less. In some embodiments, the anti-glycated BTLA antibodies provided herein have a K _D of 10 nM or less. In some embodiments, the anti-glycated BTLA antibodies provided herein have a K _D of 5 nM or less. In some embodiments, the anti-glycated BTLA antibodies provided herein have a K _D of 2 nM or less. In some embodiments, the anti-glycated BTLA antibodies provided herein have a K _D of 1 nM or less. In some embodiments, the anti-glycated BTLA antibodies provided herein have a K _D of 0.5 nM or less. In some embodiments, the anti-glycated BTLA antibodies provided herein have a K _D of 0.1 nM or less.

In some embodiments, the anti-glycated BTLA antibodies provided herein can block or neutralize the activity of BTLA. The anti-glycated BTLA antibodies can be neutralizing antibodies. The neutralizing antibodies can block the binding of BTLA to a natural ligand, such as HVEM, and can inhibit signaling pathways mediated by BTLA and/or other physiological activities thereof. The IC50 of the neutralizing antibody can be in the range of 0.01 - 10 μg/ml in a neutralizing assay (e.g., ELISA). The IC50 of the neutralizing antibody can be less than or equal to 10 μg/ml. The IC50 of the neutralizing antibody can be less than or equal to 8 μg/ml. The IC50 of the neutralizing antibody can be less than or equal to 6 μg/ml. The IC50 of the neutralizing antibody can be less than or equal to 4 μg/ml. The IC50 of the neutralizing antibody can be less than or equal to 2 μg/ml. The IC50 of the neutralizing antibody may be 1 μg/ml or less. The IC50 of the neutralizing antibody may be 0.8 μg/ml or less. The IC50 of the neutralizing antibody may be 0.6 μg/ml or less. The IC50 of the neutralizing antibody may be 0.4 μg/ml or less. The IC50 of the neutralizing antibody may be 0.2 μg/ml or less. The IC50 of the neutralizing antibody may be 0.1 μg/ml or less. The IC50 of the neutralizing antibody may be 0.08 μg/ml or less. The IC50 of the neutralizing antibody may be 0.06 μg/ml or less. The IC50 of the neutralizing antibody may be 0.04 μg/ml or less. The IC50 of the neutralizing antibody may be 0.02 μg/ml or less. The IC50 of the neutralizing antibody may be 0.01 μg/ml or less.

In some embodiments, the anti-glycated BTLA antibody provided herein specifically binds to glycated BTLA. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N75, N94, N110 or any combination thereof. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at position N75. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at position N94. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at position N110. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N75 and N94. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N75 and N110. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N94 and N110. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N75, N94, and N110.

The anti-glycated BTLA antibodies provided in the present invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the foregoing. Non-limiting examples of functional fragments include single-chain Fv (scFv) (including, for example, monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab) ₂ fragments, F(ab') ₂ fragments, disulfide-linked Fv (sdFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.

Specifically, the anti-glycated BTLA antibodies provided in the present invention include immunoglobulin molecules and molecules containing an immunologically active portion of an immunoglobulin molecule, e.g., an antigen-binding fragment that specifically binds to BTLA or glycosylated BTLA. The immunoglobulin molecules provided in the present invention can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

The anti-glycated BTLA antibodies provided herein can be monospecific, bispecific, trispecific or even multispecific. Multispecific antibodies can be specific for different epitopes of BTLA disclosed herein or can be specific for the BTLA polypeptide as well as a heterologous epitope, e.g., a heterologous polypeptide or a solid support material. In specific embodiments, the antibodies provided herein are monospecific for a given epitope of a BTLA polypeptide and do not bind to other epitopes.

By known means and as disclosed herein, polyclonal or monoclonal antibodies, antigen-binding fragments, and binding domains and CDRs (including engineered forms of any of the foregoing) specific for glycosylated BTLA, one or more of its respective epitopes, or conjugates of any of the foregoing can be produced, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds.

Antibodies can be produced from any animal source, including birds and mammals. In some embodiments, the antibodies are from sheep, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken. Additionally, newer techniques allow for the development and screening of human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression techniques allow for the production of specific antibodies in the absence of animal immunization, as disclosed in U.S. Pat. No. 6,946,546, which is incorporated herein by reference in its entirety. These techniques are described in Marks et al., Bio/Technol ., 10:779-783 (1992); Stemmer, Nature , 370:389-391 (1994); Gram et al. , Proc. Natl. Acad. Sci. USA , 89:3576-3580 (1992); Barbas et al. , Proc. Natl. Acad. Sci. USA , 91:3809-3813 (1994); and Schier et al. , Gene , 169(2):147-155 (1996), which are herein incorporated by reference in their entireties.

Methods for producing polyclonal antibodies from a variety of animal species, and for producing various types of monoclonal antibodies, including humanized, chimeric, and fully human, are well known in the art. For example, the following U.S. patents provide working descriptions of such methods and are herein incorporated by reference: U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; Nos. 5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; 6,891,024; 7,407,659; and 8,178,098.

In some embodiments, the anti-glycated BTLA antibody may be a monoclonal antibody. In some embodiments, the anti-glycated BTLA antibody may be a polyclonal antibody. An animal may be immunized with an antigen, such as a glycosylated BTLA polypeptide, to produce antibodies specific for the glycosylated BTLA polypeptide. Often, the antigen is linked or conjugated to another molecule to enhance the immune response. The conjugate may be any peptide, polypeptide, protein, or nonproteinaceous substance that is linked to the antigen used to elicit an immune response in the animal. Antibodies produced in the animal in response to the antigen immunization have multiple, non-identical molecules (polyclonal antibodies) made from multiple individual antibody-producing B lymphocytes. Given the precise conditions for polyclonal antibody production in the animal, most of the antibodies in the animal's serum recognize collective epitopes on the antigenic compound to which the animal has been immunized.

This specificity can be further improved by affinity purification to select only antibodies that recognize the antigen or epitope of interest. The process for producing monoclonal antibodies (MAbs) can begin in the same manner as the process for producing polyclonal antibodies. In some embodiments, rodents, such as mice and rats, are used in the production of monoclonal antibodies. In some embodiments, rabbit, sheep or frog cells are used in the production of monoclonal antibodies. The use of rats is well known and may offer certain advantages. Mice (e.g., BALB/c mice) are routinely used and generally provide a high percentage of stable fusions.

Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with a glycosylated BTLA polypeptide with an immortalized myeloma cell (usually a mouse myeloma). This technology provides a way to propagate a single antibody-producing cell for an infinite number of generations, thereby producing an infinite amount of structurally identical antibodies (monoclonal antibodies) having the same antigen or epitope specificity.

Anti-glycosylated BTLA antibodies can be produced by any method known in the art useful for producing polypeptides, such as in vitro synthesis, recombinant DNA production, etc. Humanized antibodies can be produced by recombinant DNA technology. The antibodies disclosed herein can also be produced using recombinant immunoglobulin expression technology. Recombinant production of immunoglobulin molecules, including humanized antibodies, is disclosed in U.S. Pat. No. 4,816,397 to Boss et al. , U.S. Pat. Nos. 6,331,415 and 4,816,567 (both to Cabilly et al. ), British Patent No. 2,188,638 to Winter et al. , and British Patent GB 2,209,757; which are incorporated herein by reference in their entireties. Techniques for recombinant expression of immunoglobulins, including humanized immunoglobulins, are also disclosed in Goeddel et al. , Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody Engineering , WH Freeman (1992); which are incorporated herein by reference in their entireties. Additional information on the production, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies , Academic Press, San Diego (1993).

Many methods have been developed to replace the light and heavy chain constant domains of a monoclonal antibody with similar domains of human origin, leaving the variable region of the foreign antibody intact. Alternatively, fully human monoclonal antibodies are produced in mice or rats that are transgenic for human immunoglobulin genes. Methods have also been developed to convert the variable domains of a monoclonal antibody to a more human form by recombinantly producing antibody variable domains that have both rodent and human amino acid sequences. In humanized monoclonal antibodies, only the hypervariable CDRs are derived from nonhuman (e.g., mouse, rat, chicken, llama) monoclonal antibodies, and the framework regions are derived from human amino acid sequences. Substituting amino acid sequences characteristic of rodent antibodies for amino acid sequences found at corresponding positions in human antibodies is thought to reduce the likelihood of a deleterious immune response during therapeutic use. Hybridomas or other cells that produce antibodies may also undergo genetic mutations or other changes that may or may not alter the binding specificity of the antibody produced by the hybridoma.

Engineered antibodies can be produced by using recombinant DNA technology with monoclonal and other antibodies to produce other antibodies or chimeric molecules that retain the antigen or epitope specificity of the original antibody, i.e., the molecule has a binding domain. Such techniques can involve introducing DNA encoding the immunoglobulin variable region or CDR of the antibody into the genetic material for the framework region, constant region, or constant region plus framework region of a different antibody. See, e.g., U.S. Patent Nos. 5,091,513 and 6,881,557, which are incorporated herein by reference.

In some embodiments, the anti-glycated BTLA antibodies are human antibodies. Human antibodies can be produced by a variety of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences (see, U.S. Pat. Nos. 4,444,887 and 4,716,111; and International Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741). Human antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made nonfunctional separately or simultaneously with the introduction of human immunoglobulin gene loci by homologous recombination. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized using conventional methods utilizing a selected antigen, e.g., all or part of a glycosylated BTLA polypeptide. Monoclonal antibodies directed against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. Pat. No. 5,916,771). The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, subsequently undergoing class switching and somatic mutation. Thus, using such technology, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol . 13:65-93, which is incorporated herein by reference in its entirety). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publications WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are herein incorporated by reference in their entireties. Additionally, companies such as Abgenix, Inc. (Fremont, CA) and Medarex (Princeton, NJ) may be involved in providing human antibodies directed against selected antigens using technologies similar to those described above.

In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen binding sequence from a non-human donor grafted onto a heterologous non-human, human or humanized sequence (e.g., framework and/or constant domain sequences). In one embodiment, the non-human donor is rat. In one embodiment, the antigen binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display screening of a human phage library, etc.). In one embodiment, a chimeric antibody provided herein has a murine V region and a human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain. In one embodiment, the murine heavy chain V region is fused to a human IgG1 C region.

Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al. , BioTechniques 4:214 (1986); Gillies et al. , J. Immunol. Methods 125:191-202 (1989); and U.S. Pat. Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397; all of which are incorporated herein by reference in their entireties. Chimeric antibodies comprising one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule can be prepared by, for example, CDR-grafting (EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al. , Protein Engineering 7:805 (1994); and Roguska et al. , Proc. Natl. Acad. Sci. USA 91:969 (1994)), and chain shuffling. shuffling) (U.S. Patent No. 5,565,332), all of which are incorporated herein by reference in their entireties.

An exemplary method for producing a recombinant chimeric anti-glycosylated BTLA antibody can comprise: a) constructing, by conventional molecular biology methods, an expression vector encoding and expressing an antibody heavy chain in which the CDRs and variable regions of a murine anti-glycosylated BTLA monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for expression of the chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector encoding and expressing an antibody light chain of the murine anti-glycosylated BTLA monoclonal antibody, thereby producing a vector for expression of the chimeric antibody light chain; c) delivering the expression vector to a host cell by conventional molecular biology methods to produce a transfected host cell for expression of the chimeric antibody; and d) culturing the transfected cell by conventional cell culture techniques to produce the chimeric antibody.

An exemplary method for producing a recombinant humanized anti-glycosylated BTLA antibody can comprise: a) constructing, by conventional molecular biology methods, an expression vector encoding and expressing an antibody heavy chain, wherein at least a portion of the variable region framework necessary to retain the CDRs and donor antibody binding specificity is derived from a non-human immunoglobulin, such as a murine anti-glycosylated BTLA monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expression of the humanized antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector encoding and expressing an antibody light chain, wherein at least a portion of the variable region framework necessary to retain the CDRs and donor antibody binding specificity is derived from a non-human immunoglobulin, such as a murine anti-glycosylated BTLA monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expression of the humanized antibody light chain; c) delivering the expression vector to a host cell, by conventional molecular biology methods, to produce a transfected host cell for expression of the humanized antibody; And d) producing humanized antibodies by culturing cells transfected by conventional cell culture techniques.

In connection with the exemplary method, the host cells may be cotransfected with such expression vectors, which preferably are identical except for the heavy and light chain coding sequences, but which may contain different selectable markers. This procedure provides identical expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both the heavy and light chain polypeptides may be utilized. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA or both. The host cell used to express the recombinant antibody may be a bacterial cell, such as Escherichia coli , or more preferably a eukaryotic cell (e.g., Chinese hamster ovary (CHO) cell or HEK-293 cell). The choice of expression vector will depend on the choice of the host cell, and may be selected to have the desired expression and regulatory characteristics in the selected host cell. Other cell lines that may be used include, but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, The Netherlands). Additionally, codon usage can be optimized when the host cell is selected to enhance protein expression, which is responsible for species-specific codon usage bias. For example, DNA encoding an antibody for expression in CHO cells may include codons preferentially used by Cricetulus griseus ( from which Chinese hamster ovary cells are derived). Codon optimization methods can be used to promote improved expression by a desired host cell (see, e.g., Wohlgemuth, et al. , Philos. Trans. R. Soc. Lond. B Biol. Sci. 366(1580):2979-2986 (2011); Jestin, et al. , J. Mol. Evol. 69(5):452-457 (2009); Bollenbach, et al. , Genome Res. 17(4):401-404(2007); Kurland, et al. , Prog. Nucleic Acid Res. Mol. Biol. 31:191-219 (1984); Grosjean, et al. , Gene 18(3): 199-209(1982)).

In one embodiment, the antibody is an immunoglobulin single variable domain derived from a camel antibody, preferably a heavy chain camel antibody without a light chain, known as a V _H H domain sequence or Nanobodies ^TM . Nanobodies ^TM (Nb) are the smallest functional fragment or single variable domain (V _H H) of a naturally occurring single chain antibody and are known to those skilled in the art. They are derived from a heavy chain-only antibody found in camels (Hamers-Casterman et al ., Nature 363: 446-448 (1993); Desmyter et al . , Nat. Struct. Biol. , 803-811 (1996)). In the "Camelidae", immunoglobulins without a light chain polypeptide are found. "Camelidae" includes Old World camels ( Camelus bactrianus and Camelus dromedarius ) and New World camels (e.g., Lama paccos , Lama glama , Lama guanicoe , and Lama vicugna ). Single variable domain heavy chain antibodies are referred to herein as nanobody ^TM or V _H H antibodies. The small size and unique biophysical properties of Nbs outperform conventional antibody fragments for recognition of uncommon or cryptic epitopes and for binding into cavities or active sites of protein targets. Furthermore, Nbs can be designed as multi-specific and multivalent antibodies, attached to reporter molecules, or humanized. Nbs are stable, survive the gastrointestinal tract, and can be readily manufactured.

When two antigen-binding sites of different specificities are combined into a single construct, bispecific antibodies have great potential as therapeutics because they have the ability to bind two different antigens with exquisite specificity. Bispecific antibodies can be produced by fusing two hybridomas, each of which is capable of producing a different immunoglobulin. Bispecific antibodies can also be produced by linking two scFv antibody fragments while omitting the Fc portion present in whole immunoglobulins. Each scFv unit in such a construct can consist of one variable domain from each of the antibody heavy (VH) and light (VL) chains, linked to each other via a synthetic polypeptide linker, the linker often being genetically engineered to be as resistant to proteolysis as possible while being as minimally immunogenic as possible. The individual scFv units can be linked by a number of techniques, including the inclusion of a short (usually less than 10 amino acids) polypeptide spacer that bridges the two scFv units to produce a bispecific single-chain antibody. The bispecific single-chain antibodies thus generated are species containing two VH/VL pairs of different specificities on a single polypeptide chain, wherein the VH and VL domains within each scFv unit are separated by a polypeptide linker that is sufficiently long to allow intramolecular association between these two domains, and the scFv units thus formed are linked to each other adjacently via a polypeptide spacer that is sufficiently short to prevent unwanted association, for example, between the VH domain of one scFv unit and the VL domain of another scFv unit.

Examples of antigen binding fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a "Fd" fragment consisting of the VH and CH1 domains; (iii) a "Fv" fragment consisting of the VL and VH domains of a single antibody; (iv) a "dAb" fragment consisting of a VH domain; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule ("scFv") in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) a bispecific single-chain Fv dimer (see U.S. Pat. No. 5,091,513); and (ix) diabodies, which are multivalent or multispecific fragments made by genetic fusion (see U.S. Patent Application Publication No. 20050214860). Fv, scFv, or diabody molecules can be stabilized by the inclusion of a disulfide bridge linking the VH and VL domains. Minibodies with scFv linked to a CH3 domain can also be made (Hu et al. , Cancer Res ., 56:3055-3061(1996)).

Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. , Cell Mol. Biol. , 49:209-216 (2003) disclose "antibody-like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have the desired advantages of a longer serum half-life as well as a less cumbersome synthetic method.

7.2.2. Glycosylated BTLA polypeptide

In another further embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated, and the polypeptide is formulated in a pharmaceutically acceptable carrier.

In some embodiments, the present invention also provides a polypeptide of at least 7 consecutive amino acids of human BTLA having at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated. In some embodiments, the polypeptide has at least 7 consecutive amino acids of human BTLA having an amino acid corresponding to glycosylated position N75. In some embodiments, the polypeptide has at least 7 consecutive amino acids of human BTLA having an amino acid corresponding to glycosylated position N94. In some embodiments, the polypeptide has at least 7 consecutive amino acids of human BTLA having an amino acid corresponding to glycosylated position N110.

For example, the polypeptide can be a fragment of amino acids 70-76 of human BTLA, wherein N75 is glycosylated. As another example, the polypeptide can be a fragment of amino acids 90-100 of human BTLA, wherein N94 is glycosylated. As yet another example, the polypeptide can be a fragment of amino acids 90-115 of human BTLA, wherein N94 and N110 are glycosylated. Those skilled in the art will appreciate that the polypeptides contemplated by the present invention include any and all polypeptides having at least seven contiguous amino acids of human BTLA, comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated.

In some embodiments, the polypeptide has at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids of human BTLA. In some embodiments, the polypeptide has at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270, 280 consecutive amino acids of human BTLA. In some embodiments, the invention provides a composition comprising at least two polypeptides provided herein. The at least two polypeptides can be separate molecules or can be linked as one molecule. In some embodiments, the composition has at least three polypeptides, at least four polypeptides, or at least five polypeptides. In some embodiments, the composition has two polypeptides, three polypeptides, four polypeptides, or five polypeptides.

In some embodiments, the polypeptides provided herein comprise a non-natural amino acid. In some embodiments, the non-natural amino acid is methylated at the α-amino group to produce a peptide having a methylated backbone. In some embodiments, the non-natural amino acid is an R-amino acid. In some embodiments, the non-natural amino acid can comprise a dye (e.g., a fluorescent dye) or an affinity tag. In some embodiments, the polypeptides provided herein comprise a chemical modification. Chemical modifications include, for example, chemical modifications using biotin, fluorescent dyes. Those skilled in the art will recognize that methods for introducing non-natural amino acids into polypeptides and chemically modifying polypeptides are well known in the art.

In some embodiments, the polypeptide of the embodiment is fused or conjugated to an immunogenic polypeptide (e.g., keyhole limpet hemocyanin, KLH). In some embodiments, the polypeptide further comprises a Cys residue at the C- or N-terminus. For example, in some embodiments, the polypeptide is conjugated to the immunogenic polypeptide by a disulfide bond at the Cys residue.

In yet another further embodiment, the invention provides an immunogenic composition comprising a polypeptide comprising a fragment of at least 7 contiguous amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated, wherein the polypeptide is formulated in a pharmaceutically acceptable carrier. In some embodiments, in some aspects, the immunogenic composition further comprises an adjuvant, such as alum or Freund's adjuvant.

In some embodiments, a method of making an antibody is provided, comprising administering to an animal a polypeptide and isolating an antibody from the animal, wherein the polypeptide has a fragment of at least 7 consecutive amino acids of human BTLA having at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated. The animal can be a mouse, a rat, a rabbit, or a human. In some aspects, the method further comprises identifying CDRs of the antibody and humanizing sequences surrounding the CDRs to produce a humanized antibody. In a further aspect, the method comprises recombinantly expressing the humanized antibody. Accordingly, in a further embodiment, an isolated antibody produced by the method described above is provided. Thus, in some embodiments, the invention provides an isolated antibody that selectively binds to a polypeptide of the invention (e.g., a fragment of at least 7 contiguous amino acids of human BTLA comprising at least one amino acid corresponding to position N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to position N75, N94, or N110 of human BTLA is glycosylated) as compared to non-glycosylated BTLA.

The polypeptides provided herein can be prepared by any method known in the art. For example, the polypeptides can be prepared by chemical synthesis or recombinant production. Exemplary methods for expressing and purifying recombinant polypeptides can be found in, for example, Scopes RK, Protein Purification - Principles and Practice, Springer Advanced Texts in Chemistry , 3 ^rd Edition (1994); Simpson RJ et al. , Basic Methods in Protein Purification and Analysis: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1 ^st Edition (2008); Green MR and Sambrook J., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 4 ^st Edition (2012); Jensen KJ et al. , Peptide Synthesis and Applications (Methods in Molecular Biology), Humana Press, 2 ^nd Edition (2013). Chemical synthesis of polypeptides can be accomplished using methods well known in the art (Kelley and Winkler, 1990, In: Genetic Engineering Principles and Methods , Setlow J. K, ed., Plenum Press, NY, Vol. 12, pp 1-19; Stewart et al ., 1984, JM Young, JD, Solid Phase Peptide Synthesis , Pierce Chemical Co., Rockford, Ill; Marglin and Merrifield, Ann. Rev. Biochem , 39:841-866, at 862 (1970). Merrifield, RB, 1963, J. Am. Chern. Soc. 85:2149-2154; Chemical Approaches to the Synthesis of Peptides and Proteins , Williams et al. , Eds., 1997, CRC Press, Boca Raton Fla.; Solid Phase Peptide See Synthesis: A Practical Approach , Atherton & Sheppard, Eds., 1989, IRL Press, Oxford, England; see also U.S. Patents 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

7.2.3. Transformations and Derivatives

Antibodies to glycated BTLA can have the ability to neutralize or counteract the effects of glycated BTLA, regardless of the animal species, monoclonal cell line, or other source of the antibody. Some animal species may be more likely to cause allergic reactions due to activation of the complement system via the Fc portion of the antibody, and therefore may be less desirable for the production of therapeutic antibodies. However, whole antibodies can be enzymatically cleaved into Fc (complement binding) fragments and antibody fragments having binding domains or CDRs. Removal of the Fc portion reduces the likelihood that the antibody fragments will cause undesirable immune responses, and thus antibodies lacking the Fc can be used for prophylactic or therapeutic treatment. As described above, antibodies can also be engineered to be chimeric, partially or fully human, to reduce or eliminate adverse immunological consequences resulting from administration to an animal of antibodies produced in or having sequences from other species.

The binding properties of anti-glycated BTLA antibodies can be further improved by screening for variants that exhibit the desired properties. For example, such improvements can be made using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles containing polynucleotide sequences encoding them. In specific embodiments, such phage can be used to display antigen-binding fragments, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing antigen-binding fragments that bind to an antigen of interest can be selected or identified using antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and M13. The antigen-binding fragments are expressed as proteins recombinantly fused to phage gene III or gene VIII proteins. Examples of phage display methods that can be used to make the antibodies or polypeptides disclosed herein include Brinkman et al. , J Immunol Methods, 182:41-50 (1995); Ames et al. , J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al. , Eur. J. Immunol. , 24:952-958(1994); Persic et al. , Gene, 187:9-18 (1997); Burton et al. , Adv. Immunol. 57:191-280 (1994); PCT Publication Nos. WO 92/001047; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743, and 5,969,108, which are herein incorporated by reference in their entireties.

As disclosed in the above references, after phage selection, the antibody coding region from the phage can be isolated and utilized to produce whole antibodies, including humanized antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described in detail below. Techniques for recombinantly producing Fab, Fab′, and F(ab′) ₂ fragments can also be utilized using methods known in the art, such as those disclosed in PCT Publication No. WO 92/22324; Mullinax, RL et al. , BioTechniques, 12(6):864-869 (1992); and Sawai et al. , Am. J. Reprod. Immunol. 34:26-34 (1995); and Better, M. et al. Science 240:1041-1043 (1988); all of which are incorporated herein by reference in their entireties. Examples of techniques that can be used to produce single chain Fvs and antibodies include those disclosed in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston, JS et al. , Methods in Enzymology 203:46-88(1991); Shu, L. et al. , Proc. Natl. Acad. Sci. (USA) 90:7995-7999; and Skerra. A. et al. , Science 240:1038-1040 (1988); all of which are incorporated herein by reference in their entireties.

Phage display technology can be used to increase the affinity of the anti-glycated BTLA antibodies disclosed herein. This technology can be used to obtain high affinity antibodies that can be used in the combinatorial methods disclosed herein. This technique, called affinity maturation, uses mutagenesis or CDR walking and re-selection using such receptors or ligands (or their extracellular domains) or antigenic fragments thereof to identify antibodies that bind to an antigen with higher affinity than the initial or parent antibody (see, e.g., Glaser, SM et al. , J. Immunol. 149:3903-3913 (1992)). Mutagenesis of entire codons, rather than single nucleotides, results in a semi-randomized repertoire of amino acid mutations. The library can be constructed to consist of a population of mutant clones, each differing by a single amino acid change in a single CDR and containing mutants representing each possible amino acid substitution for each CDR residue. Mutants with increased binding affinity for the antigen can be screened for by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity for the antigen (e.g., ELISA) (see, e.g., Wu, H. et al. , Proc. Natl. Acad. Sci. (USA) 95(11):6037-6042 (1998); Yelton, DE et al. , J. Immunol. 155:1994-2004 (1995)). CDR walking to randomize the light chain can also be used (see Schier et al. , J. Mol. Biol. 263:551-567 (1996)).

Random mutagenesis can be used in conjunction with phage display methods to identify improved CDR and/or variable regions. Phage display techniques can alternatively be used to increase (or decrease) CDR affinity by directed mutagenesis (e.g., affinity maturation or "CDR-walking"). This technique uses a target antigen or antigenic fragment to identify antibodies with CDRs that bind the antigen with higher (or lower) affinity than the initial or parent antibody (see, e.g., Glaser, SM et al. , J. Immunol. 149:3903-3913 (1992)).

Methods for achieving such affinity maturation are described, for example, in Krause, JC et al. , MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10(2011); Kuan, CT et al. , Int. J. Cancer 10.1002/ijc.25645; Hackel, BJ et al. , J. Mol. Biol. 401(1):84-96(2010); Montgomery, DL et al. , MAbs 1(5):462-474(2009); Gustchina, E. et al. , Virology 393(1):112-119 (2009); Finlay, WJ et al. , J. Mol. Biol. 388(3):541-558 (2009); Bostrom, J. et al. , Methods Mol. Biol. 525:353-376 (2009); Steidl, S. et al. , Mol. Immunol. 46(1):135-144 (2008); and Barderas, R. et al. , Proc. Natl. Acad. Sci. (USA) 105(26):9029-9034 (2008); all of which are herein incorporated by reference in their entireties.

The present invention also provides derivatives of anti-glycated BTLA antibodies or glycosylated BTLA polypeptides having one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids may be glycosylated (e.g., For example, the modified carbohydrate modifications can be (e.g., having contents such as modified mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. In some embodiments, the modified carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, facilitation of antibody assembly, conformational integrity, and antibody-mediated effector function. In some embodiments, the modified carbohydrate modifications enhance antibody-mediated effector function compared to an antibody without the carbohydrate modification. Carbohydrate modifications that lead to altered antibody-mediated effector functions are well known in the art (see, e.g., Shields, RL et al. , J. Biol. Chem. 277(30): 26733-26740 (2002); Davies J. et al. Biotechnology & Bioengineering 74(4): 288-294 (2001); all of which are incorporated herein by reference in their entireties). Methods for altering the carbohydrate content are known to those of skill in the art and include, e.g., Wallick, SC et al. , J. Exp. Med. 168(3): 1099-1109 (1988); Tao, MH et al. , J. Immunol. 143(8): 2595-2601 (1989); Routledge, EG et al. , Transplantation 60(8):847-53 (1995); See Elliott, S. et al. , Nature Biotechnol. 21:414-21(2003); Shields, RL et al. , J. Biol. Chem. 277(30): 26733-26740 (2002); all of which are incorporated herein by reference in their entireties.

Substitution variants may contain the exchange of one amino acid for another amino acid at one or more sites within the antibody or polypeptide provided herein, and may be designed to modulate one or more properties of the antibody or polypeptide, with or without loss of other functions or properties. The substitutions may be conservative, i.e., one amino acid is replaced with an amino acid of similar shape and charge. Conservative substitutions are well known in the art, and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; Methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitutions may be nonconservative, which affects the function or activity of the polypeptide. Nonconservative changes typically involve substituting a chemically different residue, such as a polar or charged amino acid, for a nonpolar or uncharged amino acid, or vice versa.

In some embodiments, the humanized antibody is a derivative antibody. Such humanized antibodies comprise amino acid residue substitutions, deletions, or additions in one or more non-human CDRs. The humanized antibody derivative can have substantially the same binding, better binding, or worse binding as compared to the non-derivative humanized antibody. In some embodiments, one, two, three, four, or five amino acid residues of a CDR are mutated, such as substituted, deleted, or added.

In some embodiments, the polypeptide is a derivative polypeptide. Such a polypeptide comprises amino acid residue substitutions, deletions or additions compared to wild-type human BTLA. The derivative polypeptide can have substantially the same binding, better binding or worse binding to an anti-glycosylated BTLA antibody compared to the non-derivative polypeptide. In some embodiments, one, two, three, four, or five amino acid residues of human BTLA are mutated, such as by substitution, deletion or addition.

The antibodies or polypeptides disclosed herein can be modified by chemical modification using techniques known to those of skill in the art, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In one embodiment, the derivative polypeptide or derivative antibody possesses a similar or identical function to the parent polypeptide or antibody. In another embodiment, the derivative polypeptide or derivative antibody exhibits an altered activity relative to the parent polypeptide or parent antibody. For example, the derivative antibody (or fragment thereof) can bind its epitope more tightly or be more resistant to proteolysis than the parent antibody.

The substitutions, additions or deletions in the derivatized antibody may be in the Fc region of the antibody and thereby serve to modify the binding affinity of the antibody to one or more FcγRs. Methods of modifying antibodies having altered binding to one or more FcγRs are known in the art, see, for example, PCT Publications WO 04/029207, WO 04/029092, WO 04/028564, WO 99/58572, WO 99/51642, WO 98/23289, WO 89/07142, WO 88/07089, and U.S. Pat. Nos. 5,843,597 and 5,642,821; all of which are incorporated herein by reference in their entireties. In some embodiments, the antibody or other molecule can have altered affinity for an activating FcγR, e.g., FcγRIIIA. Preferably, such modifications also have altered Fc-mediated effector function. Modifications that affect Fc-mediated effector function are well known in the art (see, e.g., U.S. Pat. No. 6,194,551, and WO 00/42072). In some embodiments, the modification of the Fc region results in an antibody having altered antibody-mediated effector function, altered binding to another Fc receptor (e.g., an Fc activating receptor), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity activity (CDC), phagocytic activity, or any combination thereof.

The derivative antibody or polypeptide may also have an altered half-life (e.g., serum half-life) of the parent molecule or antibody in a mammal, preferably a human. In some embodiments, such an alteration results in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life of the humanized antibody or polypeptide in a mammal, preferably a human, results in a higher serum titer of the antibody or polypeptide in the mammal, thus reducing the frequency of administration of the antibody or polypeptide and/or reducing the concentration of the antibody or polypeptide administered. Antibodies or polypeptides with increased in vivo half-lives can be produced by techniques known to those of skill in the art. For example, antibodies or polypeptides having increased in vivo half-life can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor. The humanized antibodies disclosed herein can be engineered to have increased biological half-life (see, e.g., U.S. Patent No. 6,277,375). For example, the humanized antibodies disclosed herein can be engineered in the Fc-hinge domain to have increased in vivo or serum half-life.

Antibodies or polypeptides disclosed herein having increased in vivo half-lives can be produced by attaching polymer molecules, such as high molecular weight polyethylene glycol (PEG), to the antibody or polypeptide. PEG can be attached to the antibody or polypeptide with or without a multifunctional linker, either through site-specific conjugation of PEG to the N- or C-terminus of the molecule or antibody, or through an epsilon-amino group present on a lysine residue. Linear or branched polymer derivatizations that result in minimal loss of biological activity can be utilized. The extent of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate, for example, by size exclusion or ion-exchange chromatography.

The antibodies or polypeptides disclosed herein can also be modified by the methods and coupling agents disclosed by Davis et al. (see U.S. Pat. No. 4,179,337) to provide compositions that can be injected into the mammalian circulation substantially without an immunogenic response. Since removal of the Fc portion can reduce the potential for the antibody fragment to elicit an undesirable immune response, antibodies lacking the Fc can be utilized for prophylactic or therapeutic treatments. As noted above, the antibodies can also be made chimeric, partially or fully human, to reduce or eliminate adverse immunological consequences resulting from administering to an animal antibodies produced in or having sequences from another species.

7.2.4. Fusions and Conjugates

The anti-glycated BTLA antibody or glycosylated BTLA polypeptide provided in the present invention can also be expressed as a fusion protein with another protein or chemically conjugated to another moiety.

In some embodiments, the invention provides antibodies or polypeptides having an Fc portion, wherein the Fc portion can be varied by isotype or subclass, can be chimeric or hybrid, and/or can be modified, for example, to improve effector function, control half-life, tissue accessibility, biophysical properties, e.g., increase stability, and improve production efficiency (and lower cost). Many modifications useful in the production of the disclosed fusion proteins and methods for making them are known in the art, see, e.g., Mueller, JP et al. , Mol. Immun. 34(6):441-452 (1997), Swann, PG, Curr. Opin. Immun. 20:493-499 (2008), and Presta, LG, Curr. Opin. Immun. 20:460-470 (2008). In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, e.g., a chimera with an IgG2/IgG4 Fc constant region. Modifications to the Fc region include, but are not limited to, an IgG4 modified to prevent binding to an Fc gamma receptor and complement, an IgG1 modified to improve binding to one or more Fc gamma receptors, an IgG1 modified (by amino acid changes) to minimize effector function, an IgG1 with altered/glycan-free residues (typically due to changes in the expression host), and an IgG1 with altered pH-dependent binding to FcRn. The Fc region may comprise the entire hinge region, or less than the entire hinge region.

Other embodiments include IgG2-4 hybrids and IgG4 mutants having reduced binding to FcRs and thus increased half-life. Representative IgG2-4 hybrids and IgG4 mutants are disclosed in Angal et al. , Molec. Immunol. 30(1):105-108 (1993); Mueller et al. , Mol. Immun. 34(6):441-452 (1997); and U.S. Pat. No. 6,982,323; all of which are incorporated herein by reference in their entireties. In some embodiments, the IgG1 and/or IgG2 domains are deleted, for example, Angal et al. disclose IgG1 and IgG2 wherein serine 241 is substituted with proline.

In some embodiments, the invention provides a fusion protein or polypeptide having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.

In some embodiments, the invention provides an anti-glycated BTLA antibody or a glycosylated BTLA polypeptide that is linked to, covalently bound to, or formed into a complex with at least one moiety. Such moiety may be, but is not limited to, one that increases the efficacy of the molecule as a diagnostic or therapeutic agent. In some embodiments, the moiety may be an imaging agent, a toxin, a therapeutic enzyme, an antibiotic, a radiolabeled nucleotide, or the like.

The molecules provided herein can comprise a therapeutic moiety (or one or more therapeutic moieties). The molecules provided herein can be antibodies conjugated or recombinantly fused to a therapeutic moiety, such as a cytotoxic or cytostatic agent, a therapeutic agent or a radioactive metal ion, such as an alpha-emitter. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Therapeutic moieties include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine); Alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisdichlorodiamine platinum(II) (DDP), and cisplatin); anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); Antibiotics (e.g., actinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)); Auristatin molecules (e.g., auristatin PHE, auristatin F, monomethyl auristatin E, bryostatin 1, and solastatin 10; see Woyke et al. , Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al. , Antimicrob. Agents Chemother. 45:3580-4 (2001), Mohammad et al. , Anticancer Drugs 12:735-40 (2001), Wall et al. , Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad et al. , Int. J. Oncol. 15:367-72 (1999)), all of which are incorporated herein by reference); Hormones (e.g., glucocorticoids, progestins, androgens, and estrogens), DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al. , Clin Cancer Res. 8(7):2167-76 (2002)); cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, Doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof and U.S. Patent Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, Nos. 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459); farnesyl transferase inhibitors (e.g., R115777, BMS-214662, and compounds disclosed in, e.g., U.S. Pat. Nos. 6,458,935, 6,451,812, 6,440,974, No. 6,436,960, No. 6,432,959, No. 6,420,387, No. 6,414,145, No. 6,410,541, No. 6,410,539, No. 6,403,581, No. 6,399,615, No. 6,387,905, No. 6,372,747, No. 6,369,034, No. 6,362,188, No. 6,342,765, No. 6,342,487, No. 6,300,501, No. 6,268,363, No. 6,265,422, No. 6,248,756, No. 6,239,140, No. 6,232,338, No. 6,228,865, No. 6,228,856, No. 6,225,322, No. 6,218,406, No. 6,211,193, No. 6,187,786, No. 6,169,096, No. 6,159,984, No. 6,143,766, No. 6,133,303, No. 6,127,366, No. 6,124,465, No. 6,124,295, No. 6,103,723, No. 6,093,737, No. 6,090,948, No. 6,080,870, No. 6,077,853, No. 6,071,935, No. 6,066,738, Nos. 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305); topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022; TAN-1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and rebeccamycin); bulgarein; DNA minor groove binders, for example, Hoescht dye 33342 and Hoescht dye 33258; nitidine; fagaronine; epiberberine; coralyne; beta-lapachone; BC-4-1; bisphosphonates (for example, alendronate, cimadronte, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate); HMG-CoA reductase inhibitors (e.g., lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statin, cerivastatin, lescol, lupitol, rosuvastatin, and atorvastatin); antisense oligonucleotides (e.g., U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) and pharmaceutically acceptable salts, solvates, clathrates and prodrugs thereof.

Additionally, the molecules provided in the present invention may be antibodies conjugated or recombinantly fused to a therapeutic moiety or drug moiety that modifies a given biological response. The therapeutic moiety or drug moiety should not be construed as being limited to traditional chemical therapeutics. For example, the drug moiety may be a protein, peptide, or polypeptide possessing the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; Proteins such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents such as TNF-γ, TNF-γ, AIM I (see International Publication No. WO 97/33899), AIM II (see International Publication No. WO 97/34911), Fas ligand (Takahashi et al. , 1994, J. Immunol., 6:1567-1574), and VEGF (see International Publication No. WO 99/23105), anti-angiogenic agents such as angiostatin, endostatin or components of the coagulation pathway (e.g., tissue factor); or a biological response modifier, for example, a lymphokine (e.g., interferon gamma, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin-7 ("IL-7"), interleukin-9 ("IL-9"), interleukin-10 ("IL-10"), interleukin-12 ("IL-12"), interleukin-15 ("IL-15"), interleukin-23 ("IL-23"), granulocyte-macrophage colony stimulating factor ("GM-CSF") and granulocyte colony stimulating factor ("G-CSF")), or a growth factor (e.g., growth hormone ("GH")), or a coagulant (e.g., calcium, vitamin K, tissue factor, for example, but not limited to, Hageman's factor (factor) XII), high molecular weight kininogen (HMWK), prekallikrein (PK), coagulation proteins-factor II (prothrombin), factors V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipids and fibrin monomers.

Additionally, the antibodies provided herein can be conjugated to macrocyclic chelators useful for conjugating radioactive metal ions, for example, therapeutic moieties such as alpha-emitters, for example, ²¹³ Bi, or radioactive metal ions including but not limited to ¹³¹ In, ¹³¹ LU, ¹³¹ Y, ¹³¹ Ho, ¹³¹ Sm, to the polypeptide. In some embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in detail in Denardo et al. , 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al. , 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al. , 1999, Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

The therapeutic moiety or drug conjugated or recombinantly fused to the antibody provided herein that immunospecifically binds to BTLA should be selected so as to achieve the desired prophylactic or therapeutic effect(s). In some embodiments, the antibody is a modified antibody. When deciding which therapeutic moiety or drug to conjugate or recombinantly fuse to the antibody provided herein, a physician or other medical professional should consider the following: the nature of the disease, the severity of the disease, and the condition of the individual.

In some embodiments, the moiety is an enzyme, a hormone, a cell surface receptor, a toxin (e.g., abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or schizont antiviral protein), a protein (e.g., tumor necrosis factor, an interferon (e.g., α-interferon, β-interferon), nerve growth factor, platelet derived growth factor, tissue plasminogen activator, or an apoptotic agent (e.g., tumor necrosis factor-α, tumor necrosis factor-β)), a biological response modifier (e.g., a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6")), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or macrophage colony stimulating factor ("M-CSF")), or growth factors (e.g., growth hormone ("GH")), cytotoxins (e.g., cytostatic or cytotoxic agents, e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE; e.g., vedotin) and puromycin and analogs or homologs thereof), antimetabolites (e.g., , methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, BiCNU® (carmustine; BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisdichlorodiamine platinum(II) (DDP), cisplatin); anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); These may be antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), or antimitotic agents (e.g., vincristine and vinblastine).

Techniques for conjugating such therapeutic moieties to antibodies are well known; see, e.g., Amon et al. , " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al. , " Antibodies For Drug Delivery ", in CONTROLLED DRUG DELIVERY (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ", in MONOCLONAL ANTIBODIES '84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. (eds.), 1985, pp. 475-506); “ Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy “, in MONOCLONAL ANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; Thorpe et al. , Immunol. Rev. 62:119-158 (1982); Carter et al. , Cancer J. 14(3):154-169 (2008); Alley et al. , Curr. Opin. Chem. Biol . 14(4):529-537 (2010); Carter et al. , Amer. Assoc. Cancer Res. Education. Book. 2005(1):147-154 (2005); Carter et al. , Cancer J. 14(3):154-169 (2008); Chari , Acc. Chem Res. 41(1):98-107 (2008); Doronina et al. , Nat. Biotechnology. 21(7):778-784 (2003); Ducry et al. , Bioconjug Chem. 21(1):5-13(2010); Senter, Curr. Opin. Chem. Biol. 13(3):235-244 (2009); and Teicher, Curr Cancer Drug Targets. 9(8):982-1004 (2009).

In some embodiments, the antibodies and polypeptides disclosed herein can be conjugated to a marker, such as a peptide, to facilitate purification. In some embodiments, the marker is a hexa-histidine peptide, a hemagglutinin "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson, IA et al. , Cell , 37:767-778 (1984)), or a "flag" tag (Knappik, A. et al. , Biotechniques 17(4):754-761 (1994)).

In some embodiments, the moiety can be an imaging agent that can be detected in the assay. Such an imaging agent can be an enzyme, a prosthetic group, a radiolabel, a nonradioactive paramagnetic metal ion, a hapten, a fluorescent label, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a bioluminescent molecule, a photophilic molecule, a colored particle or a ligand, for example, biotin.

In some embodiments, the enzyme includes but is not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; the prosthetic group complex includes but is not limited to streptavidin/biotin and avidin/biotin; the fluorescent material includes but is not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; the luminescent material includes but is not limited to luminol; and the bioluminescent material includes but is not limited to luciferase, luciferin and aequorin; Radioactive substances include bismuth ( ²¹³ Bi), carbon ( ¹⁴ C), chromium ( ⁵¹ Cr), cobalt ( ⁵⁷ Co), fluorine ( ¹⁸ F), gadolinium ( ¹⁵³ Gd, ¹⁵⁹ Gd), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), germanium ( ⁶⁸ Ge), holmium ( ¹⁶⁶ Ho), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In, ¹¹¹ In), iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), lanthanum ( ¹⁴⁰ La), lutetium ( ¹⁷⁷ Lu), manganese ( ⁵⁴ Mn), molybdenum ( ⁹⁹ Mo), palladium ( ¹⁰³ Pd), phosphorus ( ³² P), praseodymium ( ¹⁴² Pr), promethium ( ¹⁴⁹ Pm), rhenium ( ¹⁸⁶ Re, ¹⁸⁸ Re), rhodium ( ¹⁰⁵ Rh), lutetium ( ⁹⁷ Ru), samarium ( ¹⁵³ Sm), scandium ( ⁴⁷ Sc), selenium ( ⁷⁵ Se), strontium ( ⁸⁵ Sr), sulfur ( ³⁵ S), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), tin ( ¹¹³ Sn, ¹¹⁷ Sn), tritium ( ³ H), xenon ( ¹³³ Xe), ytterbium ( ¹⁶⁹ Yb, ¹⁷⁵ Yb), yttrium ( ⁹⁰ Y), zinc ( ⁶⁵ Zn); positron-emitting metals using various positron emission tomography techniques, and nonradioactive paramagnetic metal ions, but are not limited thereto.

Imaging agents can be conjugated to the antibodies or polypeptides disclosed herein, either directly or indirectly via an intermediate (e.g., a linker known in the art), using techniques known in the art. For example, see U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to the antibodies and other molecules disclosed herein for use as diagnostic agents. Some conjugation methods involve the use of metal chelate complexes using organic chelating agents such as, for example, diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3-6α-diphenylglycouryl-3 attached to the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers can be prepared in the presence of these coupling agents or by reaction with isothiocyanates.

In some embodiments, the antibodies or polypeptides disclosed herein can be conjugated to a second antibody to form an antibody heteroconjugate as disclosed by Segal in U.S. Patent No. 4,676,980. Such heteroconjugate antibodies may additionally bind to a hapten (e.g., fluorescein), or to a cell marker (e.g., 4-1-BB, B7-H4, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, ICOS, B7-H3, B7-H7, B7-H7CR, CD70, CD47), or to a cytokine (e.g., IL-7, IL-15, IL-12, IL-4 TGF-beta, IL-10, IL-17, IFNγ, Flt3, BLys) or a chemokine (e.g., CCL21).

In some embodiments, the anti-glycated BTLA antibodies or glycosylated BTLA polypeptides disclosed herein can also be attached to a solid support, which can be useful for immunoassays or purification of target antigens or other molecules capable of binding to the target antigen immobilized on the support via binding to the antibody or antigen-binding fragment disclosed herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

7.2.5. Protein purification

Techniques for purifying proteins are well known to those skilled in the art. These techniques involve, at one level, the homogenization and fractionation of cells, tissues or organs into polypeptide and non-polypeptide fractions. The protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity), unless otherwise specified. Analytical methods particularly suitable for the preparation of pure peptides are ion-exchange chromatography, size-exclusion chromatography, reversed-phase chromatography, hydroxyapatite chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. A particularly efficient method for purifying peptides is fast-performance liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC). As is generally known in the art, it is contemplated that the order in which the various purification steps are performed may be varied, or that some steps may be omitted, and still result in a suitable method for the production of substantially purified polypeptides.

A purified polypeptide is intended to refer to a composition that is separable from other components, wherein the polypeptide is purified to some extent relative to its naturally-occurring state. Thus, an isolated or purified polypeptide also refers to a polypeptide that is not in the environment in which it would naturally occur. Generally, "purified" will refer to a polypeptide composition that has undergone fractionation to remove various other components and wherein the composition substantially retains its expressed biological activity. When the term "substantially purified" is used, the term will refer to a composition in which the polypeptide forms the major component of the composition, for example, constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.

A variety of methods for quantifying the degree of purification of a polypeptide are known to those skilled in the art in light of the present invention. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptide in a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it to the specific activity of the initial extract, and calculate the purity therein, as assessed by the "fold purification number." The actual units used to express the amount of activity will of course depend on the specific analytical technique chosen to follow the purification, and whether or not the expressed polypeptide exhibits detectable activity.

There is no general requirement that a polypeptide always be provided in its most purified state. In fact, it is contemplated that less substantially purified products may be useful in some embodiments. Partial purification may be achieved by combining fewer purification steps or by using different forms of the same general purification scheme. For example, it is understood that cation-exchange column chromatography performed using an HPLC apparatus will generally result in a greater "fold" of purification than the same technique using a low pressure chromatography system. Methods that exhibit a lower degree of relative purification may have advantages in overall recovery of the protein product, or in maintaining the activity of the expressed protein.

Affinity chromatography is a chromatographic procedure that relies on the specific affinity between the substance to be separated and the molecule to which it can specifically bind. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then capable of specifically adsorbing the substance from solution. Elution occurs by changing the conditions to conditions where binding does not occur (e.g., changing pH, ionic strength, temperature, etc.). The matrix must be a material that does not adsorb the molecule to any significant degree and has a wide range of chemical, physical, and thermal stability. The ligand must be coupled in a manner that does not affect its binding properties. The ligand must also provide a relatively tight bond. It must be possible to elute the substance without destroying the sample or the ligand.

Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in a solution are separated based on their size, or more technically, their hydrodynamic volume. It is commonly applied to large molecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography, as opposed to the name gel permeation chromatography, which is used when an organic solvent is used as the mobile phase. The basic principle of SEC is that particles of different sizes will elute (filter) through the stationary phase at different rates. This results in the separation of particle solutions based on size. If all particles are loaded at the same time or nearly at the same time, particles of the same size will elute together.

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography often used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC uses a column containing chromatographic packing material (stationary phase), a pump to move the mobile phase(s) through the column, and a detector that measures the retention time of the molecules. The retention time varies depending on the interaction between the stationary phase, the molecule being analyzed, and the solvent(s) used.

The present invention also provides a method of assessing BTLA glycosylation, N-linked glycosylation or N-glycosylation comprising contacting a BTLA-containing sample with an antibody of the embodiment (e.g., an antibody that selectively binds to glycosylated BTLA over non-glycosylated BTLA). In some embodiments, the method is an in vitro method. In some embodiments, the sample is a cell sample.

7.2.6. Nucleic acids.

The present invention also contemplates nucleic acid molecules (DNA or RNA) encoding any of the anti-glycated BTLA antibodies or glycosylated BTLA polypeptides disclosed herein. The present invention also provides vector molecules (e.g., plasmids) capable of delivering or replicating such nucleic acid molecules. The nucleic acids can be single-stranded, double-stranded, and can contain both single-stranded and double-stranded portions.

7.3. Pharmaceutical preparations

When clinical applications of pharmaceutical compositions containing antibodies are made, it will generally be advantageous to prepare pharmaceutical or therapeutic compositions suitable for the intended application. In general, the pharmaceutical compositions may have an effective amount of an anti-glycated BTLA antibody or a glycosylated BTLA polypeptide disclosed herein, or may have additional agents dissolved or dispersed in a pharmaceutically acceptable carrier.

The present invention also provides compositions having an anti-glycated BTLA antibody or a glycosylated BTLA polypeptide disclosed herein. In some embodiments, the composition can have at least 0.1% by weight of the antibody or polypeptide. In some embodiments, the composition can have at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more by weight of the anti-glycated BTLA antibody or the glycosylated BTLA polypeptide. In other embodiments, for example, the anti-glycated BTLA or glycated BTLA polypeptide can comprise from about 2% to about 75%, from about 25% to about 60%, from about 30% to about 50%, or any range therein, of the composition's weight. The amount of active compound(s) in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained from any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations can be taken into consideration by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising a VH or VL domain of murine monoclonal antibody STC613 disclosed in Table 2. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising a VH or VL domain of murine monoclonal antibody STC626 disclosed in Table 4. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising a VH or VL domain of murine monoclonal antibody STC635 disclosed in Table 6.

In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising both the VH and VL domains of murine monoclonal antibody STC613 disclosed in Table 2. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising both the VH and VL domains of murine monoclonal antibody STC626 disclosed in Table 4. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising both the VH and VL domains of murine monoclonal antibody STC635 disclosed in Table 6.

In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising one or more VH CDRs having an amino acid sequence of any one of the VH CDRs of murine monoclonal antibody STC613 disclosed in Table 3. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising one or more VH CDRs having an amino acid sequence of any one of the VH CDRs of murine monoclonal antibody STC626 disclosed in Table 5. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising one or more VH CDRs having an amino acid sequence of any one of the VH CDRs of murine monoclonal antibody STC635 disclosed in Table 7.

In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising one or more VL CDRs having an amino acid sequence of any one of the VL CDRs of murine monoclonal antibody STC613 disclosed in Table 3. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising one or more VL CDRs having an amino acid sequence of any one of the VL CDRs of murine monoclonal antibody STC626 disclosed in Table 5. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising one or more VL CDRs having an amino acid sequence of any one of the VL CDRs of murine monoclonal antibody STC635 disclosed in Table 7.

In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising at least one VH CDR and at least one VL CDR of murine monoclonal antibody STC613 disclosed in Table 3. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising at least one VH CDR and at least one VL CDR of murine monoclonal antibody STC626 disclosed in Table 5. In some embodiments, the pharmaceutical composition can have an anti-glycosylated BTLA antibody comprising at least one VH CDR and at least one VL CDR of murine monoclonal antibody STC635 disclosed in Table 7.

In some embodiments, the pharmaceutical composition can have an anti-glycated BTLA antibody that competitively blocks (e.g., in a dose-dependent manner) a BTLA epitope disclosed herein. The BTLA epitope can be an epitope of STC613 disclosed herein. In some embodiments, the pharmaceutical composition can have an anti-glycated BTLA antibody that specifically binds to an epitope of BTLA disclosed herein. The BTLA epitope can be an epitope of STC613 disclosed herein. In some embodiments, the BTLA epitope has at least 5 consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169 .

The composition can be a pharmaceutical composition having an anti-glycated BTLA antibody or a glycosylated BTLA polypeptide as an active ingredient and a pharmaceutically acceptable carrier. The pharmaceutical composition can further comprise one or more additional active ingredients. A pharmaceutically acceptable carrier can be a carrier approved by a regulatory agency of the Federal or a state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeia for use in animals, and more specifically in humans.

As used herein, and unless otherwise specified, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, stabilizer, or vehicle with which the therapeutic agent is administered. A "pharmaceutically acceptable carrier" is a carrier that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed, and can be a sterile liquid, such as water, and an oil of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A pharmaceutically acceptable molecular entity or composition does not produce any deleterious, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. Formulations of pharmaceutical compositions with antibodies or additional active ingredients are known to those skilled in the art in light of the present invention, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference. Additionally, it will be understood that for animal (e.g., human) administration, formulations should meet standards for sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biological Standards.

The composition is contemplated to comprise from about 0.001 mg to about 10 mg total antibody or polypeptide per ml. Thus, the concentration of antibody or polypeptide in the composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any derivable range therein). Of the double, about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an anti-glycosylated BTLA antibody or a glycosylated BTLA polypeptide.

The preparation of pharmaceutical compositions having an antibody or other polypeptide disclosed herein as an active ingredient is known to those skilled in the art in light of the present invention, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference. It is also understood that for animal (including human) administration, formulations should meet sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

Pharmaceutically acceptable carriers include liquid, semi-solid, i.e., paste, or solid carriers. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. Pharmaceutically acceptable carriers are known to those skilled in the art, and include aqueous solvents (e.g., water, alcoholic/aqueous solutions, ethanol, saline, parenteral vehicles, e.g., sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters, e.g., ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delaying agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids, e.g., glycine and lysine, carbohydrates, e.g., (dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.), gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, the like and combinations thereof. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effect of the composition contained therein, its use in the administrable compositions for use in the practice of the present methods is suitable. The pH and the precise concentrations of the various ingredients in the pharmaceutical composition are adjusted according to well-known parameters. According to some embodiments of the present invention, the composition may be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, grinding, and the like. Such procedures are routine to those skilled in the art.

In some embodiments, the pharmaceutically acceptable carrier can be an aqueous pH buffered solution. Examples include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN ^™ , polyethylene glycol (PEG), and PLURONICS ^™ .

In some embodiments, the pharmaceutically acceptable carrier can be a sterile liquid, such as water and an oil of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water can be a carrier, particularly when the pharmaceutical composition is to be administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, polysorbate-80, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

Some embodiments of the present invention may have different types of carriers depending on whether it is administered in solid, liquid or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The compositions may be formulated for administration intravenously, intradermally, transdermally, intratracheally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, transmucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, directly in a lipid composition (e.g., liposomes), through a catheter, by lavage, by localized perfusion bathing of target cells, or by other methods or any combination of the foregoing known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference). Typically, such compositions may be prepared as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions upon addition of a liquid prior to injection may also be prepared; the formulations may also be emulsified.

The anti-glycated BTLA antibody or glycated BTLA polypeptide can be formulated into the composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the proteinaceous composition or with inorganic acids, for example, hydrochloric acid or phosphoric acid, or with organic acids, for example, acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide; or from organic bases, for example, isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine or procaine.

In a further embodiment, the present invention provides a pharmaceutical composition comprising a lipid. Lipids can broadly include a class of substances that are characteristically insoluble in water and extractable by organic solvents. Examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., designed or produced by humans). Lipids can be biological. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids having ether- and ester-linked fatty acids, polymeric lipids, and combinations thereof. Other compounds than those specifically disclosed herein that are understood by those skilled in the art to be lipids may also be used.

Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid vehicles. For example, the antibody or polypeptide may be dispersed in a solution containing a lipid, dissolved in a lipid, emulsified in a lipid, mixed with a lipid, combined with a lipid, covalently linked to a lipid, contained as a suspension in a lipid, contained or complexed in micelles or liposomes, or otherwise associated with a lipid or lipid structure, by any means known to those skilled in the art. The dispersion may or may not result in the formation of liposomes.

Typically, the components of the composition are supplied separately or mixed together in unit dosage form as a dry lyophilized powder or anhydrous concentrate in a sealed container, such as an ampoule or sachette, indicating the quantity of active agent. If the composition is to be administered by infusion, the composition may be provided with an infusion vial containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water or saline for injection may be provided so that the components can be mixed prior to administration.

The amount of active ingredient in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained from any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into consideration by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

A unit dose or dosage means a physically distinct unit suitable for use in a subject, each unit containing a predetermined quantity of the pharmaceutical composition calculated to produce the desired response as described above in conjunction with its administration, i.e., an appropriate route and regimen of treatment. The amount to be administered, depending on the frequency of treatment and the unit dose, will depend on the desired effect. The actual dosage of the composition of the present embodiments administered to a patient or subject may be determined by physical and physiological factors such as the body weight, age, health and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or coexisting therapeutic interventions, the idiopathic condition of the patient, the route of administration, and the efficacy, safety and toxicity of the specific therapeutic agent. In other non-limiting examples, the dosage can range from about 1 microgram/kg/body weight per administration, to about 5 micrograms/kg/body weight, to about 10 micrograms/kg/body weight, to about 50 micrograms/kg/body weight, to about 100 micrograms/kg/body weight, to about 200 micrograms/kg/body weight, to about 350 micrograms/kg/body weight, to about 500 micrograms/kg/body weight, to about 1 milligram/kg/body weight, to about 5 milligrams/kg/body weight, to about 10 milligrams/kg/body weight, to about 50 milligrams/kg/body weight, to about 100 milligrams/kg/body weight, to about 200 milligrams/kg/body weight, to about 350 milligrams/kg/body weight, to about 500 milligrams/kg/body weight, to about 1000 milligrams/kg/body weight or more, and any derivable range therein. In non-limiting examples of ranges derivable from the numbers enumerated herein, ranges of from about 5 milligrams/kg/body weight to about 100 milligrams/kg/body weight, from about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, etc. may be administered based on the numbers disclosed above. In any case, the practitioner responsible for administration shall be responsible for the concentration of the active ingredient(s) in the composition. and will determine the appropriate capacity(s) for the individual entity.

Those skilled in the art will appreciate that the compositions disclosed herein are not limited by the specific nature of the therapeutic agent. For example, such compositions may be provided in a formulation with physiologically acceptable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic agents may be administered to mammals for veterinary use, such as in livestock, and for clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary depending on the specific needs of the individual subject, as well as the type and mode of administration. The actual dosage of the composition administered to an animal patient, including a human patient, may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or coexisting therapeutic interventions, idiopathic symptoms of the patient, and route of administration. Depending on the dosage and route of administration, the desired dosage and/or frequency of administration of the effective amount may vary depending on the response of the subject. In any event, the practitioner responsible for administration shall be responsible for the concentration of the active ingredient(s) in the composition. and will determine the appropriate capacity(s) for the individual entity.

7.4. Treatment of disease

As used herein, and unless otherwise specified, the term "subject" refers to an animal that is the subject of treatment, observation, and/or experimentation. "Animal" includes vertebrates and invertebrates, such as fish, shellfish, reptiles, birds, and especially mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, apes, and humans. In some embodiments, the subject is a human.

As used herein, and unless otherwise specified, the term "cancer" or "cancerous" refers to a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, blood cancers and solid tumors.

As used herein, and unless otherwise specified, the terms "treat," "treating," or "treatment" refer to the performance of a procedure or modality in a subject, or the administration or application of a therapeutic agent to a subject, for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, treatment may comprise administering to the subject a therapeutically effective amount of an anti-glycated BTLA antibody. When used in reference to a cancer patient, the terms "treat," "treating," or "treatment" refer to the action of potentially reducing the severity of a cancer, delaying or slowing the progression of a cancer, including (a) inhibiting the growth of a cancer, decreasing the rate at which the cancer grows, stopping the development of a cancer, reducing the invasiveness of the cancer, or preventing the metastasis of the cancer, and (b) causing regression of the cancer, delaying or minimizing one or more symptoms associated with the presence of the cancer, or prolonging the survival of a cancer patient.

As used herein, and unless otherwise specified, the term "therapeutically effective amount" refers to an amount of an agent (e.g., an antibody or polypeptide disclosed herein or any other agent disclosed herein) sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder, or condition, and/or symptoms associated therewith. A therapeutically effective amount of an agent, including a therapeutic agent, can be an amount necessary to (i) reduce or ameliorate the progression or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, occurrence, or onset of a given disease, disorder, or condition, and/or (iii) improve or enhance the prophylactic or therapeutic effect of another treatment (e.g., a treatment other than the administration of an antibody provided herein). The therapeutically effective amount of a substance/molecule/agent of the invention (e.g., an anti-glycated BTLA antibody) can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount includes an amount at which the therapeutically beneficial effects of a substance/molecule/agent exceed any toxic or detrimental effects.

As used herein, and unless otherwise specified, the term "administer" or "administering" refers to the act of injecting or otherwise physically delivering a substance from outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method disclosed herein or known in the art. When a disease, disorder or condition or its symptoms are being treated, the administration of the substance typically occurs after the onset of the disease, disorder or condition or its symptoms. When a disease, disorder or condition or its symptoms are being prevented, the administration of the substance typically occurs prior to the onset of the disease, disorder or condition or its symptoms.

The present invention also provides therapeutic uses of anti-glycated BTLA antibodies (e.g., STC613, STC626, or STC635) and glycosylated BTLA polypeptides. These antibodies or polypeptides can be used to modulate the activity of BTLA signaling. These antibodies or polypeptides can also be used to treat diseases by inhibiting the inhibitory activity of BTLA on T cell activation or proliferation. Accordingly, the present invention provides uses of such antibodies or polypeptides in upregulating the immune system of a subject by inhibiting or blocking BTLA signaling.

In some embodiments, the present invention also provides therapeutic uses of anti-glycated BTLA antibodies (e.g., STC613, STC626, or STC635) and glycosylated BTLA polypeptides in the treatment of cancer. Upregulation of the immune system is particularly desirable in the treatment of cancer, and therefore the present invention also provides methods for treating cancer. Cancer refers to a neoplasm or tumor resulting from the abnormal, uncontrolled growth of cells. The cancer can be a primary cancer or a metastatic cancer.

In some embodiments, the anti-glycated BTLA antibodies provided herein (e.g., STC613, STC626, or STC635 or a humanized variant thereof) can modulate an immune response in a subject. In some embodiments, the anti-glycated BTLA antibodies provided herein can promote T cell activation. In some embodiments, the anti-glycated BTLA antibodies provided herein can promote T cell proliferation. In some embodiments, the anti-glycated BTLA antibodies provided herein can increase cytokine production. In some embodiments, the anti-glycated BTLA antibodies provided herein can enhance T-cell dependent apoptosis of a cell expressing BTLA or inhibit proliferation of a cell expressing BTLA.

Accordingly, the present invention provides a method of modulating an immune response in a subject by administering an effective amount of an anti-glycated BTLA antibody provided herein (e.g., STC613, STC626, or STC635 or a humanized variant thereof). Modulating the immune response can include (a) increasing T cell activation; (b) increasing T cell proliferation; and/or (c) increasing cytokine production.

In some embodiments, the present invention provides therapeutic uses of anti-glycosylated BTLA antibodies having an antigen binding fragment comprising the VH or VL domain of murine monoclonal antibody STC613 disclosed in Table 2, murine monoclonal antibody STC626 disclosed in Table 4, or murine monoclonal antibody STC635 disclosed in Table 6. In some embodiments, the anti-glycosylated BTLA antibody can have an antigen binding fragment comprising both the VH and VL domains of murine monoclonal antibody STC613 disclosed in Table 2, murine monoclonal antibody STC626 disclosed in Table 4, or murine monoclonal antibody STC635 disclosed in Table 6. In some embodiments, the invention provides therapeutic uses of anti-glycosylated BTLA antibodies comprising at least one VH CDR having an amino acid sequence of any one of the VH CDRs of murine monoclonal antibody STC613 disclosed in Table 2, murine monoclonal antibody STC626 disclosed in Table 4, or murine monoclonal antibody STC635 disclosed in Table 6. In some embodiments, the anti-glycosylated BTLA antibodies comprise at least one VL CDR having an amino acid sequence of any one of the VL CDRs of murine monoclonal antibody STC613 disclosed in Table 2, murine monoclonal antibody STC626 disclosed in Table 4, or murine monoclonal antibody STC635 disclosed in Table 6. In some embodiments, the anti-glycated BTLA antibody comprises at least one VH CDR and at least one VL CDR of murine monoclonal antibody STC613 disclosed in Table 2, murine monoclonal antibody STC626 disclosed in Table 4, or murine monoclonal antibody STC635 disclosed in Table 6.

In some embodiments, the invention provides therapeutic uses of an anti-glycated BTLA antibody that competitively blocks (e.g., in a dose-dependent manner) a BTLA epitope disclosed herein. The BTLA epitope can be an epitope of STC613 disclosed herein. In some embodiments, the invention provides therapeutic uses of an anti-glycated BTLA antibody that specifically binds to an epitope of BTLA disclosed herein. The BTLA epitope can be an epitope of STC613 disclosed herein. In some embodiments, the BTLA epitope has at least five consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, or 169 .

The present invention also provides therapeutic uses of the anti-glycated BTLA antibodies provided herein. In some embodiments, the anti-glycated BTLA antibodies provided herein specifically bind to glycated BTLA. In some embodiments, the anti-glycated BTLA antibodies specifically bind to BTLA that is glycosylated at positions N75, N94, N110 or any combination thereof. In some embodiments, the anti-glycated BTLA antibodies specifically bind to BTLA that is glycosylated at position N75. In some embodiments, the anti-glycated BTLA antibodies specifically bind to BTLA that is glycosylated at position N94. In some embodiments, the anti-glycated BTLA antibodies specifically bind to BTLA that is glycosylated at position N110. In some embodiments, the anti-glycated BTLA antibodies specifically bind to BTLA that is glycosylated at positions N75 and N94. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N75 and N110. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N94 and N110. In some embodiments, the anti-glycated BTLA antibody specifically binds to BTLA that is glycosylated at positions N75, N94, and N110.

In some embodiments, the polypeptide or antibody of the invention (e.g., a glycosylated BTLA polypeptide or an antibody that binds to glycosylated BTLA, such as STC613, STC626, or STC635) can be administered to treat cancer. Cancers for which the present methods of treatment are useful include those found in any malignant cell type, such as a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, tumors of an organ selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of a myeloid tumor, a T or B cell malignancy, a leukemia, a lymphoma, an blastoma, a myeloma, and the like. Additional examples of cancers that can be treated using the methods provided in the present invention include carcinomas, lymphomas, blastomas, sarcomas, leukemias, squamous cell carcinomas, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, melanomas, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanomas, nodular melanomas, and B-cell lymphomas (low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade NHL). including, but not limited to, follicular NHL (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloblastic leukemia (CLL).

The cancer may be any of the following histologic types, specifically but not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphhoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial coli polyposis; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; Acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid ovarian carcinoma; adnexal carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; adenoid cell carcinoma; adenosquamous cell carcinoma; Adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma, malignant; malignant granulosa cell tumor; malignant androgenetic tumor; Sertoli cell carcinoma; malignant stromal cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytosis; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; polycystic rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; Renal blastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant brenner tumor, malignant; phyllodes tumor; synovial sarcoma; malignant mesothelioma; anaplastic germ cell tumor; embryonal carcinoma; malignant teratoma; struma ovarii, malignant; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; Kaposi sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastic tumor; ameloblastic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; Protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroblastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; Immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasmacytoid leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, the antibodies or polypeptides provided in the present invention can be used to treat cancer that is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

Polypeptides or antibodies can be used as anti-tumor agents in a variety of aspects of the present invention. In specific embodiments, the present invention provides a method of using a polypeptide or antibody as an anti-tumor agent, comprising contacting a population of tumor cells with a therapeutically effective amount of the polypeptide or antibody for a period of time sufficient to inhibit tumor cell growth.

A variety of delivery systems are also known that can be used to administer anti-glycated BTLA antibodies or related molecules, or related pharmaceutical compositions, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or fusion proteins, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987 , J. Biol. Chem. 262:4429-4432), or construction of nucleic acids as part of retroviruses or other vectors.

Methods of administration provided by the present invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes), by injection. In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided by the present invention are administered intramuscularly, intravenously, subcutaneously, intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, or intradermally. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration may also be used, for example, by use of an inhaler or nebulizer and by use of formulations utilizing an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,20; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publications WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903; all of which are incorporated herein by reference in their entireties. In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered locally to the area in need of treatment, which may be accomplished, for example, by local infusion, by injection, or by implantation, wherein the implant is a porous, nonporous, or gelatinous material, including a membrane such as a sialastic membrane, or a fiber. In some embodiments, when administering the antibodies or other molecules disclosed herein, care is taken to use a material that is not absorbed by the antibodies or other molecules.

In some embodiments, the antibodies or polypeptides provided herein are formulated as liposomes for targeted delivery. Liposomes are vesicles composed of concentrically arranged phospholipid bilayers that encapsulate an aqueous phase. Liposomes typically contain various types of lipids, phospholipids, and/or surfactants. The components of the liposome are arranged in a bilayer configuration similar to the lipid arrangement of biological membranes. Liposomes can be useful delivery vehicles, in part, because of their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and are provided herein, see, for example, Epstein et al. , 1985 , Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al. , 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; See U.S. Patent Nos. 4,485,045 and 4,544,545; all of which are incorporated herein by reference in their entireties.

The present invention also provides methods for preparing liposomes having an extended serum half-life, i.e., an improved circulation time, such as disclosed in U.S. Patent No. 5,013,556. In some embodiments, the liposomes used in the methods provided herein are not rapidly cleared from the circulation, i.e., are not taken up by the mononuclear phagocytic system (MPS). The present invention also provides sterically stable liposomes prepared using general methods known to those of skill in the art. The sterically stable liposomes can contain a lipid component having a bulky, highly flexible hydrophilic moiety, which reduces unwanted reactions of the liposomes with serum proteins, reduces opsonization with serum components, and reduces recognition by the MPS. The sterically stable liposomes can be prepared using polyethylene glycol. For the preparation of liposomes and sterically stable liposomes, see, for example, Bendas et al. , 2001 BioDrugs, 15(4): 215-224; Allen et al. , 1987 FEBS Lett. 223:42-6; Klibanov et al. , 1990 FEBS Lett., 268: 235-7; Blum et al. , 1990 , Biochim. Biophys. Acta., 1029: 91-7; Torchilin et al. , 1996 , J. Liposome Res. 6:99-116; Litzinger et al. , 1994 , Biochim. Biophys. Acta, 1190: 99-107; Maruyama et al. , 1991 , Chem. Pharm. Bull., 39: 1620-2; Klibanov et al. , 1991 , Biochim Biophys Acta, 1062; 142-8; Allen et al. , 1994 , Adv. Drug Deliv. Rev, 13: 285-309, which are herein incorporated by reference in their entireties.

The present invention also provides liposomes adapted for specific organ targeting, see, e.g., U.S. Pat. No. 4,544,545, or for specific cell targeting, see, e.g., U.S. Patent Application Publication No. 2005/0074403, which is herein incorporated by reference in its entirety. Liposomes particularly useful for use in the compositions and methods provided herein can be produced by a reverse phase evaporation process using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes can be extruded through a filter of defined pore size to produce liposomes of a desired diameter. In some embodiments, a molecule having an antigen binding fragment, e.g., F(ab′), can be conjugated to the liposome using methods previously disclosed; see, e.g., Martin et al. , 1982 , J. Biol. Chem. 257: 286-288, which is herein incorporated by reference in its entirety.

The humanized or chimeric antibodies disclosed herein may also be formulated as immunoliposomes. Immunoliposomes refer to liposome compositions in which antibodies or fragments thereof are covalently or noncovalently linked to the surface of the liposome. The chemistry of linking antibodies to the surface of the liposome is known in the art, see, for example, U.S. Pat. No. 6,787,153; Allen et al. , 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44; Hansen et al. , 1995 , Biochim. Biophys. Acta, 1239: 133-144, which are incorporated herein by reference in their entireties. In some embodiments, the immunoliposomes for use in the methods and compositions provided herein are further sterically stabilized. In some embodiments, the humanized antibodies disclosed herein are covalently or noncovalently linked to a hydrophobic anchor that is stably anchored within the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, such as phosphatidylethanolamine (PE), phosphatidylinositol (PI). Any biochemical strategy known in the art can be used to form a covalent linkage between the antibody and the hydrophobic anchor, see, for example, J. Thomas August ed., 1997, Gene Therapy: Advances in Pharmacology , Volume 40, Academic Press, San Diego, Calif., p. 399-435, which is herein incorporated by reference in its entirety. For example, a functional group on the antibody molecule can react with an active group on the liposome-associated hydrophobic anchor, for example, the amino group of a lysine side chain on the antibody can be coupled to liposome-associated N-glutaryl-phosphatidylethanolamine that has been activated with a water-soluble carbodiimide; Alternatively, the thiol group of the reduced antibody can be coupled to the liposome via a thiol-reactive anchor such as pyridylthiopropionylphosphatidylethanolamine. See, e.g., Dietrich et al. , 1996 , Biochemistry, 35: 1100-1105; Loughrey et al. , 1987 , Biochim. Biophys. Acta, 901: 157-160; Martin et al. , 1982 , J. Biol. Chem. 257: 286-288; Martin et al. , 1981 , Biochemistry, 20: 4429-38, which are herein incorporated by reference in their entireties. Immunoliposome formulations comprising anti-glycated BTLA antibodies can be particularly effective as therapeutics because they deliver the active ingredient to the cytoplasm of target cells, i.e., cells containing receptors to which the antibody binds. In some embodiments, immunoliposomes may have an increased half-life in the blood, particularly in target cells, and may be internalized into the cytoplasm of target cells, thereby avoiding loss of the therapeutic agent or degradation by the endolysosomal pathway.

The immunoliposome compositions provided herein can have one or more vesicle-forming lipids, an antibody or other molecule of the invention or a fragment or derivative thereof, and optionally, a hydrophilic polymer. The vesicle-forming lipids can be lipids having two hydrocarbon chains, such as an acyl chain, and a polar head group. Examples of vesicle-forming lipids include phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin, and glycolipids, such as cerebrosides, gangliosides. Additional lipids useful in the formulations provided herein are known to those skilled in the art and are included within the disclosure. In some embodiments, the immunoliposome compositions further comprise a hydrophilic polymer, such as polyethylene glycol, and ganglioside GM1, which increases the serum half-life of the liposomes. Methods of conjugating hydrophilic polymers to liposomes are well known in the art and are included within the disclosure. Additional exemplary immunoliposomes and methods for their preparation are described, for example, in U.S. Patent Application Publication No. 2003/0044407; PCT International Publication No. WO 97/38731, Vingerhoeads et al. , 1994 , Immunomethods, 4: 259-72; Maruyama, 2000 , Biol. Pharm. Bull. 23(7): 791-799; Abra et al. , 2002 , Journal of Liposome Research, 12(1&2): 1-3; Park, 2002 , Bioscience Reports, 22(2): 267-281; Bendas et al. , 2001 BioDrugs, 14(4): 215-224, J. Thomas August ed., 1997, Gene Therapy: Advances in Pharmacology , Volume 40, Academic Press, San Diego, Calif., p. 399-435; all of which are incorporated herein by reference in their entireties.

The present invention also provides a method of treating a cancer patient by administering to the patient a unit dose of an anti-glycated BTLA antibody. The present invention also provides a method of treating a cancer patient by administering to the patient a unit dose of a glycosylated BTLA polypeptide. A unit dose means a physically distinct unit suitable as a unitary dosage for a subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.

The antibody, polypeptide or composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount to be administered will depend on the subject being treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required to be administered will depend on the judgment of the practitioner and will be unique to each individual. However, suitable dosage ranges for systemic applications are disclosed herein and will depend on the route of administration. Suitable treatment regimens for initial and booster administration are also contemplated and typically include repeated administrations at intervals of one hour or more, with subsequent injections or other administrations following the initial administration. Exemplary multiple administrations are disclosed herein and are useful for maintaining continuously elevated serum and tissue levels of the polypeptide or antibody. Alternatively, continuous intravenous infusions sufficient to maintain blood concentrations within the ranges specified for in vivo therapy are contemplated.

The therapeutically effective dose is a predetermined amount calculated to achieve the desired effect. In general, the dosage will vary depending on the patient's age, condition, sex, and degree of disease and can be determined by a person skilled in the art. The dosage can be adjusted by an individual doctor if there are complications.

In some embodiments, the antibody, polypeptide, or pharmaceutical composition provided herein is packaged in a sealed container, such as an ampoule or sachet. In one embodiment, the antibody, polypeptide, or pharmaceutical composition provided herein is supplied as a dry sterile lyophilized powder or anhydrous concentrate in a sealed container, which may be reconstituted with water or saline to a concentration suitable for administration to a subject. In some embodiments, the antibody, polypeptide, or pharmaceutical composition provided herein is supplied as a dry sterile lyophilized powder in a sealed container in a unit dosage amount of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized antibody, polypeptide, or pharmaceutical composition provided herein should be stored at 2 to 8 ° C. in their original containers and should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour, after reconstitution. In alternative embodiments, the antibody, polypeptide, or pharmaceutical composition provided herein is supplied in liquid form in a sealed container indicating the amount and concentration of the antibody, polypeptide, or pharmaceutical composition. In some embodiments, the liquid form of the antibody, polypeptide, or pharmaceutical composition provided herein is supplied in a sealed container at at least 1 mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml.

The precise dosage to be used in the formulation will also depend on the route of administration and the severity of the condition and should be determined by the practitioner's judgment and the circumstances of each patient. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems. For anti-glycated BTLA antibodies or glycated BTLA polypeptides, the dosage administered to a patient is typically 0.01 mg/kg to 100 mg/kg of patient body weight. In some embodiments, the dosage administered to the patient is from 0.01 mg/kg to 20 mg/kg, from 0.01 mg/kg to 10 mg/kg, from 0.01 mg/kg to 5 mg/kg, from 0.01 to 2 mg/kg, from 0.01 to 1 mg/kg, from 0.01 mg/kg to 0.75 mg/kg, from 0.01 mg/kg to 0.5 mg/kg, from 0.01 mg/kg to 0.25 mg/kg, from 0.01 to 0.15 mg/kg, from 0.01 to 0.10 mg/kg, from 0.01 to 0.05 mg/kg, or from 0.01 to 0.025 mg/kg of patient body weight. Specifically, the dosage administered to the patient may be 0.2 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg. Doses as low as 0.01 mg/kg are expected to show notable pharmacodynamic effects. Dosage levels of 0.10-1 mg/kg are expected to be most appropriate. Higher doses (e.g., 1-30 mg/kg) may also be expected to be effective. In general, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to the foreign polypeptide. Therefore, lower dosages of human antibodies and less frequent administration may be achieved. In addition, the dosage and frequency of administration of the antibodies or polypeptides provided herein may be reduced by enhancing absorption and tissue penetration of the antibodies, for example, by modifications such as lipidation.

In another embodiment, the composition can be delivered in a controlled release or sustained release system. Any technique known to those skilled in the art can be used to produce a sustained release formulation comprising one or more antibodies, molecules or pharmaceutical compositions provided herein. See, for example, U.S. Patent No. 4,526,938; PCT Publication No. WO 91/05548; PCT Publication No. WO 96/20698; Ning et al. , Radiotherapy & Oncology 39:179-189 (1996), Song et al. , PDA Journal of Pharmaceutical Science & Technology 50:372-397 (1995); Cleek et al. , Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854 (1997); and Lam et al. , Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760(1997); all of which are incorporated herein by reference in their entireties. In one embodiment, the pump can be used in a release control system (see Langer, supra; Sefton, 1987 , CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al. , 1980, Surgery 88:507; and Saudek et al. , 1989 , N. Engl. J. Med. 321:574). In other embodiments, polymeric materials can be used to achieve controlled release of antibodies or polypeptides (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983 , J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. , 1985 , Science 228:190; During et al. , 1989, Ann. Neurol. 25:351; Howard et al. , 1989 , J. Neurosurg. 7 1:105); U.S. Patent Nos. 5,679,377; 5,916,597; See U.S. Patent Nos. 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253); all of which are incorporated herein by reference in their entireties.

Examples of polymers that can be utilized in controlled release formulations include, but are not limited to, poly(-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamides, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In another embodiment, the controlled release system can be positioned proximate the therapeutic target (e.g., the lung), requiring only a portion of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). In another embodiment, polymeric compositions useful as controlled release implants are disclosed in Dunn et al., which is incorporated herein by reference in its entirety. (see U.S. Patent No. 5,945,155). Based on the therapeutic effect of controlled in situ release of bioactive substances from polymer systems, implantation can generally occur anywhere within the body of a patient in need of therapeutic treatment.

In another embodiment, a non-polymeric sustained release system is utilized, wherein a non-polymeric implant within the body of an individual is utilized as the drug delivery system. Upon implantation into the body, the organic solvent of the implant will disperse, disperse, or leach from the composition into the surrounding tissue fluid, and the non-polymeric material will gradually aggregate or precipitate to form a solid microporous matrix (see U.S. Pat. No. 5,888,533). Controlled release systems are also discussed in the review by Langer (1990 , Science 249:1527-1533). Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more of the therapeutic agents provided herein. See, e.g., U.S. Pat. No. 4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698; Ning et al. , 1996 , Radiotherapy & Oncology 39:179-189; Song et al. , 1995 , PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. , 1997 , Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al. , 1997 , Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760; all of which are herein incorporated by reference in their entireties.

The invention also provides embodiments wherein the composition comprises a nucleic acid encoding an antibody or polypeptide provided in the invention, wherein the nucleic acid can be administered in vivo to promote expression of its encoded antibody or other molecule by constructing the nucleic acid as part of a suitable nucleic acid expression vector and administering the nucleic acid such that it becomes intracellular, for example, by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or by coating with lipid or cell-surface receptors or transfectants, or by linking the nucleic acid to a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al. , 1991 , Proc. Natl. Acad. Sci. USA 88 :1864-1868). Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and incorporated into the host cell DNA for expression.

Treatment of a subject using a therapeutically effective amount of an antibody, polypeptide or pharmaceutical composition provided herein may comprise a single treatment or a series of treatments. It is contemplated that the antibodies, polypeptides or pharmaceutical compositions provided herein may be administered systemically or locally to treat a disease, such as to inhibit tumor cell growth or to kill cancer cells in a cancer patient with locally advanced or metastatic cancer. They may be administered intravenously, intrathecally and/or intraperitoneally. They may be administered alone or in combination with anti-proliferative drugs. In one embodiment, they are administered to reduce the cancer load in a patient prior to surgery or other procedures. Alternatively, they may be administered after surgery to prevent residual cancer (e.g., cancer that is not removed by surgery) from surviving. In some embodiments, they may be administered after regression of a primary cancer to prevent metastasis.

7.5. combination therapy

In some embodiments, the compositions and methods of the embodiments relate to administering an antibody that selectively binds to a glycosylated BTLA polypeptide or glycosylated BTLA, in combination with a second or additional therapeutic agent. Such a therapeutic agent may be used in the treatment of any disease associated with BTLA or glycosylated BTLA. For example, the disease may be cancer, and the second therapeutic agent may be an anti-cancer or anti-hyperproliferative therapy.

Methods and compositions, including combination therapies, enhance the therapeutic or protective effects of other anticancer or anti-hyperproliferative therapies, and/or increase the therapeutic effects. The therapeutic and prophylactic methods and compositions can be provided in combined amounts effective to achieve the desired effect, such as killing cancer cells and/or inhibiting cell hyperproliferation. This process can involve administering a polypeptide or antibody and a second therapy. The second therapy may or may not have a direct cytotoxic effect. For example, the second therapy can be an agent that upregulates the immune system without having a direct cytotoxic effect. The tissue, tumor, or cell can be exposed to one or more compositions or pharmacological formulation(s) that include one or more of the agents (e.g., an antibody or an anticancer agent), or can be exposed by exposing the tissue, tumor, and/or cell to two or more distinct compositions or formulations, wherein one composition provides 1) the polypeptide or antibody, 2) the anticancer agent, or 3) both the polypeptide or antibody and the anticancer agent. Additionally, such combination therapies may be used in conjunction with chemotherapy, radiotherapy, surgery, or immunotherapy.

When applied to a cell, the terms "contacted" and "exposed" are used herein to describe the process by which a therapeutic polypeptide or antibody and a chemotherapeutic or radiotherapeutic agent are delivered to or positioned directly adjacent to a target cell. To effect cell death, for example, both agents are delivered to the cell in combined amounts effective to kill the cell or prevent cell division.

The anti-glycated BTLA antibody or glycated BTLA polypeptide can be administered prior to, during, subsequent to, or in various combinations with the second or additional anticancer treatment. The administrations can be spaced from simultaneous to several minutes to several days to several weeks. In embodiments where the antibody or polypeptide is administered to the patient separately from the anticancer agent, there will generally not be a significant period of time between the respective delivery times, such that the two compounds can still exert a beneficial combined effect on the patient. In such instances, it is contemplated that the anti-glycated BTLA antibody or glycated BTLA polypeptide and the second therapy can be administered to the patient within about 12 to 24 or 72 hours of each other, and more particularly within about 6 to 12 hours of each other. In some situations, the time for treatment can be significantly extended, such that several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration.

In some embodiments, the course of treatment can last from 1 to 90 days, including any number of days therebetween. It is contemplated that one agent can be given on any day from day 1 to day 90, including any number of days therebetween, and the other agent can be given on any day from day 1 to day 90, including any number of days therebetween, or any combination thereof. The patient can be given one or multiple doses of the agent(s) within a single (24-hour) period. It is also contemplated that there will be a period of time following the course of treatment during which no anticancer treatment is administered. This period can last from 1 to 7 days, and/or from 1 to 5 weeks, and/or from 1 to 12 months, including any number of days therebetween, depending on the patient's condition, e.g., their prognosis, severity, health, etc. The treatment cycles can be repeated as needed.

Various combinations may be used. Some examples of treatments comprising anti-glycated BTLA antibodies or glycated BTLA polypeptides as “A” and a second anticancer treatment as “B” are listed below:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration to a patient of any antibody, polypeptide or pharmaceutical composition provided herein in combination with a second therapy will follow general protocols for administration of such second therapies, taking into account the toxicity of the second therapy, if any. Accordingly, in some embodiments, there is a step of monitoring for toxicity attributable to the combination therapy.

Chemotherapy

A wide range of chemotherapeutic agents can be used as second-line therapy according to the present invention. Chemotherapeutic agents can be compounds or compositions administered in the treatment of cancer. These agents or drugs can be classified by their mode of action within the cell, for example, by whether and at what stage of the cell cycle they affect. Alternatively, agents can be characterized based on their ability to induce chromosomal and mitotic abnormalities by directly cross-linking DNA, intercalating into DNA, or affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines, and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamin; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; calystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongestatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; Antibiotics, such as enediyne antibiotics (e.g., calichemicins, especially calichemicin gamma1I and calichemicin omega1I); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicins; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; eliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethanes; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinosides ("Ara-C");cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurines; platinum coordination complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g. , CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

Radiation therapy

Another conventional anticancer treatment that may be used in combination with the methods and compositions disclosed herein is radiotherapy, or radiation therapy. Radiotherapy includes the use of γ-rays, X-rays, and/or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (see U.S. Pat. Nos. 5,760,395 and 4,870,287; all of which are incorporated herein by reference in their entireties), and UV-irradiation. All of these agents are likely to cause extensive damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance.

The tumor microenvironment is inherently suppressive due to the presence of regulatory T cells and myeloid-derived suppressor cells that infiltrate the tumor and function to suppress immune responses. In addition, the expression of some suppressive molecules on T cells and antigen-presenting cells (APCs) can limit effective immune responses. Radiation mediates anti-tumor effects through the induction of tumor cell apoptosis, senescence, and autophagy, which in some circumstances can stimulate more effective immune responses.

The abscopal effect is a physiological process by which targeted radiation to a primary tumor induces an antitumor response at distant sites not within the radiation field. The mechanism responsible for the abscopal effect is thought to be immune-mediated and involves enhanced presentation of tumor antigens to T cells and the release of cytokines and other proinflammatory factors that stimulate local and systemic immune responses. Since the abscopal effect affects tumors located distant from the primary tumor receiving radiation therapy, agents capable of inducing the abscopal effect would be particularly beneficial in the treatment of metastatic tumors, which are often more difficult to treat once they have spread to secondary sites in the body.

The anti-glycated BTLA antibodies or glycated BTLA polypeptides disclosed herein can stimulate local and systemic immune responses. In some embodiments, a therapeutically effective amount of the antibody, polypeptide or pharmaceutical composition disclosed herein is administered prior to, concurrently with or subsequent to radiotherapy to achieve a synergistic abscorbic effect.

In some embodiments, a therapeutically effective amount of an antibody, polypeptide or pharmaceutical composition disclosed herein that effectively sensitizes a tumor to irradiation in a host is administered. The irradiation may be ionizing radiation, and in particular gamma rays. In some embodiments, the gamma rays are emitted by a linear accelerator or by a radionuclide. Irradiation of a tumor with a radionuclide may be external or internal.

In some embodiments, administration of the antibody, polypeptide, or pharmaceutical composition disclosed herein is initiated at most 1 month, particularly at most 10 days or 1 week prior to irradiation of the tumor. Additionally, irradiation of the tumor is fractionated such that administration of the antibody, polypeptide, or pharmaceutical composition disclosed herein is maintained in the interval between the first and last irradiation sessions.

The investigation may also be X-ray radiation. Dose ranges for X-rays range from daily doses of 50 to 200 roentgens for extended periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the intensity and type of radiation emitted, and absorption by neoplastic cells.

Immunotherapy

Those skilled in the art will appreciate that immunotherapy may be combined or used in conjunction with the methods of the present invention. In the context of cancer therapy, immunotherapy agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. Checkpoint inhibitors, such as ipilumimab, are other such examples. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may act as the effector of the therapy, or the antibody may actually recruit other cells to cause cell death. The antibody may also be conjugated to a drug or toxin (e.g., a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin) and act solely as a targeting agent. Alternatively, the effector may be a lymphocyte bearing surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

In one embodiment of immunotherapy, the tumor cells bear some marker that is amenable to targeting, i.e., is not present on most other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative embodiment of immunotherapy is to combine an anti-cancer effect with an immunostimulatory effect. Immunostimulatory molecules are also present, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use include immune adjuvants , such as Mycobacterium bovis , Plasmodium falciparum , dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, Infect Immun . , 66(11):5329-36(1998); Christodoulides et al. , Microbiology , 66(11):5329-36(1998)); cytokine therapies, such as interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al. , Clin Cancer Res. , 4(10):2337-47 (1998); Davidson et al. , J Immunother. , 21(5):389-98(1998); Hellstrand et al ., Acta Oncol. 37(4):347-53(1998)); gene therapy, for example, TNF, IL-1, IL-2, and p53 (Qin et al. , Proc Natl Acad Sci USA, 95(24):14411-6(1998); Austin-Ward and Villaseca, Rev Med Chil , 126(7):838-45 (1998); U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-PD1, anti-PDL1, anti-CD20, anti-ganglioside GM2, and anti-p185 (Topalian et al. , The New England journal of medicine , 366:2443-2454 (2012); Brahmer et al. , The New England journal of medicine 366:2455-2465 (2012); Hollander, Front Immunol (2012): 3:3. doi: 10.3389/fimmu.2012.00003; Hanibuchi et al. , Int J Cancer , 78(4):480-5(1998); U.S. Pat. No. 5,824,311), all of which are herein incorporated by reference in their entireties. It is contemplated that one or more anticancer therapeutics may be utilized in the present disclosure involving an anti-glycated BTLA antibody or a glycosylated BTLA polypeptide.

surgery

Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and/or destroyed, and may be used in conjunction with other treatments, such as the present invention's treatment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryotherapy, electrosurgery, and microscopic-guided surgery (Mohs' surgery).

When all or part of a cancer cell, tissue, or tumor is removed, a cavity may be formed in the body. Treatment may be by perfusion, direct injection, or local application of the area with additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be in varying doses.

Other agents

It is contemplated that other agents may be used in combination with some aspects of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that cause upregulation of cell surface receptors and gap junctions, cytostatic and differentiating agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing the number of gap junctions and thereby increasing cell-to-cell signaling may increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents may be used in combination with some aspects of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the susceptibility of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the present invention to improve therapeutic efficacy.

7.6. Kits and Diagnostics

In various aspects, the present invention provides kits containing therapeutic agents and/or other therapeutic and delivery agents. In some embodiments, kits for preparing and/or administering a therapeutic agent provided herein are contemplated. The kits can include one or more sealed vials containing any of the pharmaceutical compositions provided herein. The kits can include, for example, at least an anti-glycated BTLA antibody or a glycosylated BTLA polypeptide, and reagents for preparing, formulating, and/or administering a component provided herein, or performing one or more steps of a method provided herein.

In some embodiments, the kit may comprise an anti-glycated BTLA antibody and at least one adjuvant. In some embodiments, the kit may comprise a glycosylated BTLA polypeptide and at least one adjuvant.

In some embodiments, the kit further comprises a second anticancer agent. The second anticancer agent can be a chemotherapeutic agent, an immunotherapy agent, a hormonal therapy agent, or a cytokine.

In some embodiments, the kit may also include suitable container means, such as an Eppendorf tube, an assay plate, a syringe, a bottle or a tube, which is non-reactive with the components of the kit. The container may be made of a sterilizable material, such as plastic or glass.

The kit may describe the procedural steps of the methods disclosed herein and may include instructions that follow substantially the same procedures as those disclosed herein or are known to those skilled in the art. The instructions may be in a computer-readable medium containing machine-readable instructions that, when executed by a computer, cause the display of actual or hypothetical procedures for delivering a pharmaceutically effective amount of an antibody or polypeptide provided by the invention. The kit may also include instructions in the form prescribed by a governmental agency governing the manufacture, use, or sale of pharmaceutical or biological products, which instructions reflect approval by the agency for manufacture, use, or sale for human administration.

8. Example

It is to be understood that modifications that do not substantially change the nature and spirit of the various embodiments disclosed herein are also contemplated. Accordingly, the following examples are intended to be illustrative and not limiting in any way.

Example 1 - Production of glycated BTLA-conjugated antibodies

A panel of monoclonal antibodies against recombinant glycosylated BTLA polypeptides is produced using standard techniques (e.g., by injecting polypeptides comprising glycosylated epitopes as immunogens in rats (Aurrand-Lions et al. , Immunity , 5, 391-405 (1996))). Briefly, human glycosylated BTLA polypeptide conjugated to 100 μg KLH carrier protein (keyhole limpet hemocyanin, Pierce) and mixed with adjuvant S6322 (Sigma) can be used to immunize female Wister rats. In total, three injections will be performed every 9 days. Two days after the last sc injection of human glycosylated BTLA polypeptide, blasts from the draining lymph nodes will be fused to Sp2/0 cells and hybridomas will be selected. Grown clones are screened by ELISA for the production of monoclonal antibodies that specifically recognize human glycosylated BTLA. Positive clones are subcloned, rescreened, and further tested. The antibodies are purified on a protein G-Sepharose column (GE HealthCare) according to the manufacturer's instructions. The monoclonal antibodies are used to study in vivo tumor graft models. The VH and VL chains of the antibodies are sequenced and the complementarity determining regions (CDRs) are determined by the IMGT numbering system (Lefranc et al. , Nuc Acids Res. , 27:209-212 (1999)).

As indicated above, it is desirable to utilize humanized derivatives of mouse monoclonal antibodies for certain purposes, including, for example, use in the in vivo therapy of human diseases. To form such humanized antibodies, the framework sequences of the mouse monoclonal antibodies (the "parent" sequences) are first aligned with the framework sequences of a set of "acceptor" human antibodies to identify differences in the framework sequences. Humanization is accomplished by substituting framework residues that are not matched between the parent and acceptor sequences. Substitutions at potentially important sites, such as those in the Vernier region, the VH/VL interchain interface, or within the CDR canonical class-determining sites, have been analyzed for future back mutations (see Foote, J. et al. , J. Molec. Biol. 224:487-499 (1992)).

The domain content of each amino acid chain and the approximate boundaries of each domain can be determined using the Conserved Domain Database (COD) (Marchler-Bauer, et al. (2011) Nucleic Acids Res. 39:D225-D229). The variable domain boundaries can be precisely determined along the boundaries of the CDRs according to several commonly used definitions (Kabat, EA et al. (1991) " Sequences of Proteins of Immunological Interest ," Fifth Edition. NIH Publication No. 91-3242; Chothia, C. et al. , J. Mol. Biol. 196:901-917 (1987); Honegger, A. et al. , J. Molec. Biol. 309(3):657-670 (2001)).

Multiple alignments of parental sequences to mouse and human germline sequences are generated using MAFFT (Katoh, K. et al. , Nucleic Acids Res. 30: 3059-3066 (2002)) and entries in each alignment are sorted by sequence identity to the parental sequence. The reference set is reduced to a unique set of sequences by gathering at 100% sequence identity and excluding unnecessary entries.

Selection of the optimal receptor framework is based on the overall parent antibody sequence identity to the receptor across the frameworks of the two chains; however, positions constituting the VH/VL interchain interface are of particular interest. Additionally, CDR-loop lengths and CDR positions responsible for a distinct set of canonical structures defined for five of the CDRs (Chothia, C. et al ., J. Mol. Biol. 196:901-917 (1987); Martin, AC et al. , J. Molec. Biol . 263:800-815 (1996); Al-Laziniki, B. et al. , J. Molec. Biol. 273:927-948 (1997)) are compared to the germline to determine which germline frameworks are known to have both identical interface residues and support similar CDR-loop conformations.

Based on the sequence alignment of the parent antibody to the human germline, the closest matching entry is identified. The selection of the preferred human germline is based on the following criteria in order: (1) sequence identity across the framework; (2) identical or compatible interchain interface residues; (3) support loops having a parental CDR canonical conformation; (4) a combination of heavy and light chain germlines found in the expressed antibody; and (5) the presence of N-glycosylation sites that should be removed.

A structural model of the Fv region of a humanized antibody is generated. Candidate structural template fragments for the entire Fv as well as FRs and CDRs are scored, ranked and selected from an antibody database based on their sequence identity to the target and quantitative crystallographic measures of the template structure, such as resolution in angstroms (Å).

To structurally align the CDRs to the FR template, five residues on either side of the CDR are incorporated into the CDR template. The alignment of the fragments is generated based on the overlapping segments and the resulting structural sequence alignment. The template fragments along the alignment are processed by MODELLER (SalI, A. et al.; J. Molec. Biol. 234:779-815 (1993)). This protocol generates conformational restraints derived from a set of aligned conformational templates. An ensemble of structures satisfying the constraints is generated by a conjugate gradient and simulated annealing optimization procedure. A model structure is selected from this ensemble based on the score of the protein structure and the energy score derived from satisfying the conformational constraints. The model is tested and the side chains at different positions between the target and template are optimized using a side chain optimization algorithm and the energy is minimized. A suite of visualization and computational tools are used to evaluate CDR conformational variability, local packing, and surface analysis to select one or more desirable models.

A structural model of the parent antibody is built and tested for defects such as poor atomic packing, bond length, bond angles, or variations in dihedral angles. These defects may represent potential problems in the structural stability of the antibody. The modeling protocol seeks to minimize such defects. The initial structural model of the humanized Fv contains all safe substitutions (i.e., substitutions that do not affect binding affinity or stability) and cautious substitutions (i.e., substitutions at positions that are likely to be associated with reduced binding affinity or reduced stability). Substitutions at positions that are thought to be associated with a risk of reducing binding affinity or stability are left unchanged. Template screening and selection are performed separately from the parent template screening to generate a good independent model rather than a closely matching variant model of the parent antibody. As evaluation of potential substitutions is performed, the model is updated to reflect the effects of desirable substitutions and back mutations.

Mouse monoclonal antibodies (mAbs) were generated against human glycosylated BTLA polypeptide essentially as described above. Briefly, antibody-producing hybridomas against BTLA were obtained by fusion of SP2/0 murine myeloma cells according to a standard protocol with spleen cells isolated from BALB/c mice immunized with human glycosylated BTLA polypeptide coupled to 100 μg KLH carrier protein. Prior to fusion, sera from immunized mice were verified for binding to the immunogen using FACS. T293 cells overexpressing BTLA WT (fully glycosylated) were mixed with T293 cells overexpressing fully non-glycosylated BTLA, tagged with biotin. The mixed cells were incubated with primary antibodies against BTLA and further washed with secondary antibodies conjugated with FITC. After washing, the fluorescence intensity (MFI) was measured to assess the relative binding of antibodies to membrane-bound glycosylated or non-glycosylated BTLA. Antibodies that exhibited significantly higher MFI to glycosylated BTLA than to non-glycosylated BTLA were identified as “glycospecific” antibodies. Several monoclonal antibody-producing hybridomas were produced, including 36 hybridomas producing mouse mAbs designated STC601 to STC636.

Example 2 - Isotyping of glycated BTLA-binding antibodies

The isotypes of STC601 to STC636 mAbs were determined by ELISA of hybridoma supernatants using Sigma-Aldrich ISO2 Sigma Mouse Monoclonal Antibody Isotyping Reagent.

Briefly, 100 μL hybridoma supernatant (1:20) or purified antibody (1 μg/mL) was adsorbed in white Nunc Maxisorp 96-well plates at 37°C for 1 h, washed, blocked with 1% BSA in PBS at 21°C for 30', washed, and 100 μL of isotyping solution (Sigma Aldrich, St. Louis, MO, USA) diluted 1:1000 in PBS was added at 21°C for 30', 1:5000 anti-goat IgG-HRP was added at 21°C for 15', and analyzed with BioRad Clarity Western ECL substrate (BioRad, Hercules, CA, USA) and Victor X3 (PerkinElmer, Hopkinton, MA, USA). It's happening.

Table 9 shows the isotype determination results. STC601-STC636 mAbs were found to contain IgG1, IgG2A, IgG2B, and IgG3/M isotypes.

Table 9: Isotypes of anti-BTLA mAb STC601-STC636

Example 3 - FACS analysis of glycated BTLA-binding antibodies

The ability of anti-BTLA mAbs STC601-STC636 to bind cell-surface expressed BTLA was analyzed by fluorescence-activated cell sorting (FACS).

In summary, BTLA was recombinantly overexpressed in 293T cells and the binding of STC601-STC636 to BTLA-293T cells and 293T vector control was analyzed by FACS.

Cell culture, stable transfectants, and transfection. All cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were grown in DMEM/F12 or RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). BTLA-stable transfectant 293T cells were selected using 10 μg/mL puromycin (InvivoGen, San Diego, CA). For transient transfection, cells were transfected with plasmid DNA encoding BTLA using SN liposomes (Hu, MC et al., 2004, Cell, 117:225-237), Lipofectamine 2000, Lipofectamine LTX (Life Technologies, Carlsbad, CA), or PEI.

Flow cytometry. Cells overexpressing BTLA or empty vector were dissociated by trypsinization and harvested at 2 x 10^6 cells/mL in cell staining buffer (CSB) (BioLegend, San Diego, CA, USA). 50 μL of cells were aliquoted into 96-well round-bottom plates, and 50 μL of 20 μg/mL primary antibody was added, gently mixed, and incubated for 1 h at 4 °C in the dark. Cells were washed with CSB and incubated with anti-mouse IgG-PE conjugate (10 μg/mL) with DAPI (1:100) for 30' at 21 °C in the dark. Cells were washed and data were acquired using a Guava EasyCyte HT (Millipore Darmstadt, Germany) or FACS Celesta (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer.

Table 10 shows exemplary FACS analysis results demonstrating anti-BTLA mAb binding to cell-surface expressed BTLA. Both STC601 - STC636 anti-BTLA mAbs were found to bind to cell-surface expressed BTLA, as indicated by stronger binding FACS signal (increased % Gate, increased MR) observed in BTLA expressing 293T cells compared to 293T empty vector control.

Table 10: FACS analysis of anti-BTLA mAb STC601-STC636

Example 4 - Glycospecificity of glycated BTLA-binding antibodies

The sugar specificity of anti-BTLA mAb was analyzed by dot-blot and Western blot analyses.

Dot blot

The glycospecific binding of STC601-STC636 anti-BTLA mAbs to glycated and deglycosylated (N-glycosidase F (PNGaseF) treated) BTLA was tested in dot blot analysis.

Briefly, glycated or deglycosylated BTLA (0.5 μg/well; 5 μg/mL; 100 μl/well) was immobilized onto nitrocellulose membranes in a 96-well dot blot apparatus (Bio-Dot BioRad, Hercules, CA, USA). After blocking, the membranes were incubated with hybridoma supernatant (5 μg/mL; 100 μl/well) or purified mouse monoclonal antibody (1 μg/mL; 100 μl/well) for 12 h at 4 °C. Antibody binding to immobilized BTLA on nitrocellulose membranes was detected via secondary antibody (e.g., 1:5,000 - 1:20,000 anti-mouse-HRP secondary; Jackson Labs, Bar Harbor, ME, USA) and imaging (e.g., SuperSignal West Femto, ThermoFisher, Waltham, MA, USA or Chemdoc imager, Bio-Rad, Hercules, CA, USA).

Figures 1a and 1b show the results of exemplary dot blot analyses of anti-BTLA mAbs STC601 - STC636. Each anti-BTLA mAb (0.5 μg/well loaded) was tested for binding to glycosylated BTLA (PNGaseF "-") or deglycosylated BTLA (PNGaseF "+"). A nonspecific antibody control ("IgG", 0.25 μg/well loaded) and a commercially available BTLA reference antibody (BioLegend, San Diego, CA, USA, "BioLegend", 5 μg/well loaded) were also included in the analyses. Figure 1a shows the experimental arrangement of test antibodies and controls. Figure 1b depicts the results of experimental dot blot analyses obtained with STC601 - STC636 mAbs and controls. Several monoclonal antibodies, including STC602, STC604, STC605, STC606, STC607, STC608, STC609, STC610, STC611, STC612, STC613, STC614, STC616, STC617, STC618, STC619, STC620, STC621, STC622, STC623, STC624, STC626, STC627, STC628, STC629, STC630, STC631, STC632, STC634, STC635, and STC636, exhibited glycospecific binding to glycosylated BTLA.

Western Blot

The glycospecificity of anti-BTLA mAb was further analyzed by immunoprecipitation/Western blot analysis.

Immunoblot analysis. Immunoblot analysis was performed as previously described (Lim et al., 2008, Gastroenterology, 135:2I 28-40; and Lee et al., 2007, Cell, 130:440-455). Image acquisition and quantification of band intensities were performed using a ChemDoc imager (Bio-Rad, Hercules, CA, USA). Secondary antibodies were anti-mouse or -rabbit Alexa Fluor 488 or 594, and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA).

Figure 2 shows exemplary Western blot analysis results for anti-BTLA mAbs STC604, STC605, STC606, STC608, STC610, STC613, STC618, STC622, STC626, STC627, STC628, STC630, STC635, and STC636. In the Western blot analysis, anti-BTLA mAbs were tested for their ability to recognize fully glycosylated wild-type BTLA and several BTLA mutants lacking two (N75/2NQ, N94/2NQ, N116/2NQ) or all three (3NQ) of the N-glycosylation sites of BTLA. All tested antibodies were found to recognize glycosylated wild-type BTLA (WT). Additionally, all tested anti-BTLA mAbs were found to recognize at least one BTLA double mutant harboring only a single glycosylation site at BLTA N75, N94, or N110. In general, the band intensities shown in the Western blots of Figure 2 represent the specific glycosylation motif recognized by each of the tested antibodies and the binding strength of the tested antibody to the recognized glycosylation motif. In contrast, all tested anti-BTLA mAbs were found to exhibit only background binding, if any, to the BTLA triple mutant harboring none of the N75, N94, or N110 glycosylation sites of BTLA.

Example 5 - Binding affinity and binning of glycated BTLA-binding antibodies

Anti - BTLA mAbs, including STC601-STC636, were characterized for their respective binding affinities for BTLA and further evaluated in competition binding assays and epitope binning experiments.

KD determination and binning. Antibody/BTLA complexes in 20 nM solutions were loaded onto the APC sensor from ForteBio and a baseline was established in PBS (assay buffer) with 1 mg/mL BSA. Binding was performed by immersing the sensor in anti-BTLA antibody in assay buffer. Dissociation was performed in fresh assay buffer. All experiments were performed with the sensor shaking at 1,000 rpm. The data were fitted to a 1:1 binding model using ForteBio data analysis software to estimate the extent of association and dissociation. KD values were calculated using the ratio kd/ka. In a typical epitope binning assay, 10 nM BTLA-His was preincubated with αHis antibody (10 nM) for 1 h at 21°C. Control antibody (20 nM) was loaded onto the AMC sensor (ForteBio, Monlo Park, CA, USA) and the remaining Fc-binding sites on the sensor were blocked with whole mouse IgG antibody (Jackson, Bar Harbor, ME, USA). The sensors were exposed to pre-incubated antigen/secondary antibody mixtures. Raw data were processed using Data Analysis Software 7.0 (ForteBio, Monlo Park, CA, USA) and antibody pairs were evaluated for competitive binding. Additional binding by the second antibody is indicative of a non-captured epitope (non-competitor), whereas no binding is indicative of epitope blocking (competitor). SPR Biacore X-100 was also used for kD determination. Protein A chips or mouse IgG capture antibody-immobilized CM5 chips (Biacore, Žilina, Slovakia) were coated with antibodies to 600 response units (RU) and BTLA ECD was injected into the microfluidic channel. The kD values were obtained using the fitting tool of the BIAevaluation software (Biacore, Žilina, Slovakia).

Table 11 summarizes the kinetic binding constants (k _a and k _d rates) and binding affinities (K _D values) of anti-BTLA mAbs STC604, STC610, STC613, STC618, STC622, STC626, and STC635 to BTLA. Figures 3a-c illustrate the results of exemplary SPR (BIAcore ^TM ) experiments to determine the kinetic binding constants and binding affinities of anti-BTLA mAbs STC613, STC626, and STC635 to BTLA.

Table 11: Binding affinity of anti-BTLA mAbs to BTLA.

Figure 4a and Table 12 illustrate the results of exemplary competition analysis and binning experiments using STC613. Figure 4b and Table 13 illustrate the results of exemplary competition analysis and binning experiments using STC636. Table 14 provides a summary of the binning results. STC613 and STC636 were found to not compete with each other for BTLA binding. STC605, STC608, STC626, STC627, STC628, STC630, STC631, and STC636 were found to compete with STC636 for BTLA binding, but not with STC613. STC604, STC606, STC610, STC613, STC618, STC622, and STC635 were found to compete with STC613 for BTLA binding, but not with STC636.

Table 12: Binning of anti-BTLA mAbs using STC613

Table 13: Binning of anti-BTLA mAbs using STC636

Table 14: Summary of binning results

Example 6 - Neutralizing activity of glycated BTLA-binding antibodies

Anti - BLTA mAbs were evaluated using ELISA assays for their ability to inhibit BTLA-HVEM protein interaction.

Inhibition of BTLA and HVEM interaction by ELISA. Antibody neutralizing activity was assessed using the BTLA:HVEM Inhibitor Screening Assay Kit (Cat. # 72008, BPS Bioscience, San Diego, CA, USA) according to the manufacturer's instructions. BTLA was coated onto 96-well plates and then first 0.5 or 5 μg/mL antibody, followed by biotinylated HVEM, was added to the reaction, followed by addition of streptavidin-HRP. Chemiluminescence was measured after addition of HRP substrate.

Table 15 provides exemplary ELISA results demonstrating the ability of anti-BTLA mAbs to inhibit BTLA-HVEM protein interactions, expressed as % inhibition compared to the control reflecting the signal of uninhibited BTLA-HVEM interaction. For example, at a concentration of 5 μg/ml, STC613 was found to inhibit 99.0% of the BTLA-HVEM interaction, STC626 was found to inhibit 96.8% of the BTLA-HVEM interaction, and STC635 was found to inhibit 97.3% of the BTLA-HVEM interaction. Figures 5 and 6 show exemplary results of BTLA-HVEM competition ELISAs. STC613 was found to inhibit the BTLA-HVEM interaction with an IC ₅₀ of 1.088 μg/ml. STC626 was found to inhibit the BTLA-HVEM interaction with an IC ₅₀ of 0.416 μg/ml.

Table 15: Inhibition of BTLA:HVEM interaction by anti-BTLA mAbs

Example 7 - Epitope Mapping of STC613

Epitope mapping of anti-BTLA mAb STC613 was performed using chemical cross-linking, high-resolution MALDI mass spectrometry, and nLC-orbitrap mass spectrometry (CovalX AG, Zurich, Switzerland).

The BTLA antigen used for STC613 epitope mapping had the following amino acid sequence:

Table 16-20 provides the sequences of individual peptides identified after Asp-N, trypsin, chymotrypsin, elastase, and thermolysin proteolysis. In combination, the BTLA peptides identified in all five proteolytic samples cover 100% of the BTLA amino acid sequence of SEQ ID NO: 86.

Table 16 provides 34 BTLA peptides identified after trypsin proteolysis, covering 84.3% of the BTLA amino acid sequence of SEQ ID NO: 86.

Table 16: BTLA peptides identified after trypsin proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequence of SEQ ID NO: 86.

Table 17 provides 75 BTLA peptides identified after chymotrypsin proteolysis, covering 96.88% of the BTLA amino acid sequence of SEQ ID NO: 86.

Table 17: BTLA peptides identified after chymotrypsin proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequence of SEQ ID NO: 86.

Table 18 provides seven peptides identified after ASP-N proteolysis, covering 26.56% of the BTLA amino acid sequence of SEQ ID NO: 86.

Table 18: BTLA peptides identified after ASP-N proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequence of SEQ ID NO: 86.

Table 19 provides four BTLA peptides identified after elastase proteolysis, covering 21.09% of the BTLA amino acid sequence of SEQ ID NO: 86.

Table 19: BTLA peptides identified after elastase proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequence of SEQ ID NO: 86.

Table 20 provides 18 BTLA peptides identified after thermolysin proteolysis, covering 50.00% of the BTLA amino acid sequence of SEQ ID NO: 86.

Table 20: BTLA peptides identified after thermolysin proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequence of SEQ ID NO: 86.

To determine the BTLA epitope of STC613 with high resolution, the BTLA/STC613 complex was incubated with deuterated cross-linkers, followed by multienzyme digestion (Asp-N, trypsin, chymotrypsin, elastase, thermolysin) and analysis of the sample using nLC-Orbitrap mass spectrometry. Trypsin, ASP-N, and elastase digestion did not result in detection of cross-linked BTLA and STC613 peptides. Table 21 provides five cross-linked STC613 and BTLA peptides identified after chymotrypsin proteolysis. Table 22 provides one cross-linked STC613-BTLA peptide identified after thermolysin proteolysis.

Table 21: BTLA peptides identified as deuterated cross-linkers after chymotrypsin proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequences of SEQ ID NOs: 2 and 4 (protein 1) and SEQ ID NO: 86 (protein 2).

Table 22: BTLA peptides identified as deuterated cross-linkers after thermolysin proteolysis

* Peptide positions are indicated relative to the BTLA amino acid sequences of SEQ ID NOs: 2 and 4 (protein 1) and SEQ ID NO: 86 (protein 2).

Figure 7 shows a graph showing the results of a higher resolution analysis of the BTLA/STC613 molecular interface. The analysis showed that the BTLA epitope of STC613 contains three regions of BTLA, including the amino acid sequences IKRQSEHSILA (SEQ ID NO: 167), VKLEDRQTSWK (SEQ ID NO: 168), and NGSYRCSANFQ (SEQ ID NO: 169).

The BTLA epitope of STC613 was found to contain amino acids R12, H16, K51, T57, S82, and S86 of BTLA (SEQ ID NO: 86).

* * *

Throughout this application, various publications have been referenced. The entire contents of these publications are incorporated herein by reference in order to more fully disclose the art to which the present invention pertains. While certain specific embodiments of the invention have been provided herein, it will be apparent to those skilled in the art that various changes and modifications may be made. Such modifications are also intended to fall within the scope of the appended claims.

References

The following documents are specifically incorporated herein by reference to the extent they provide exemplary procedural or other details supplementary to those disclosed herein.

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U.S. Patent No. 6,861,572

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U.S. Patent No. 6,891,024

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U.S. Patent No. 7,407,659

U.S. Patent No. 8,178,098

United States Patent Application Publication No. 20050214860

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SEQUENCE LISTING <110> STCUBE, INC. <120> ANTIBODIES SPECIFIC TO GLYCOSYLATED BTLA AND METHODS OF USE THEREOF <130> 604556-228009 <140> <141> <150> 62/262,293 <151> 2015-12-02 <160> 169 <170> PatentIn version 3.5 <210> 1 <211> 289 <212> PRT <213> Homo sapiens <400> 1 Met Lys Thr Leu Pro Ala Met Leu Gly Thr Gly Lys Leu Phe Trp Val 1 5 10 15 Phe Phe Leu Ile Pro Tyr Leu Asp Ile Trp Asn Ile His Gly Lys Glu 20 25 30 Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile 35 40 45 Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr Cys Ala 50 55 60 Asn Arg Pro His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr Cys Val 65 70 75 80 Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn Ile Ser 85 90 95 Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn Gly Ser 100 105 110 Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser His Ser 115 120 125 Thr Thr Leu Tyr Val Thr Asp Val Lys Ser Ala Ser Glu Arg Pro Ser 130 135 140 Lys Asp Glu Met Ala Ser Arg Pro Trp Leu Leu Tyr Arg Leu Leu Pro 145 150 155 160 Leu Gly Gly Leu Pro Leu Leu Ile Thr Thr Cys Phe Cys Leu Phe Cys 165 170 175 Cys Leu Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala 180 185 190 Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr 195 200 205 Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly 210 215 220 Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser 225 230 235 240 Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val 245 250 255 Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu Ala 260 265 270 Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg 275 280 285 Ser <210> 2 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Arg Asp Asp 20 25 30 Tyr Val His Trp Leu Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Lys Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Val Arg Glu Gly Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 3 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 3 gaggttcagc tgcagcagtc tggggctgag cttgtgaggc caggggcctc agtcaagttg 60 tcctgcacag cttctggctt taacattaga gacgactatg tgcactggtt gaaacagagg 120 cctgatcagg gcctggagtg gattggaagg attgatcctg cgaatggtaa aactaaatat 180 gacccgaagt tccaggacaa ggccactata actgcagaca catcctccaa cacagcctac 240 ctgcagctca gcagcctgac atctgaggac actgccgtct atttctgtgt tagagagggg 300 ggtagtaact acgactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360 tca 363 <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 4 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Leu Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Ile His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 5 <211> 336 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 5 gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgca agtcaagtct gagcctctta gatagtgatg gaaagacata tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tgtgggagtt tattattgct ggcaaggtat tcattttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 6 Gly Phe Asn Ile Arg Asp Asp 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 7 Asp Pro Ala Asn Gly Lys 1 5 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 8 Glu Gly Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 9 Gly Phe Asn Ile Arg Asp Asp Tyr Val Met 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 10 Arg Ile Asp Pro Ala Asn Gly Lys Thr Lys 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 11 Glu Gly Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 12 Asp Asp Tyr Val Met 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 13 Arg Ile Asp Pro Ala Asn Gly Lys Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 14 Glu Gly Gly Ser Asn Tyr Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 15 Arg Asp Asp Tyr Val Met 1 5 <210> 16 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 16 Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Lys Thr Lys 1 5 10 <210> 17 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 17 Val Arg Glu Gly Gly Ser Asn Tyr Asp Tyr Ala Met Asp 1 5 10 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 18 Lys Ser Ser Leu Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 19 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 20 Trp Gln Gly Ile His Phe Pro Arg Thr 1 5 <210> 21 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 21 Lys Ser Ser Leu Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 22 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 23 Trp Gln Gly Ile His Phe Pro Arg Thr 1 5 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 24 Lys Ser Ser Leu Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 25 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 26 Trp Gln Gly Ile His Phe Pro Arg Thr 1 5 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 27 Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 28 Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp 1 5 10 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 29 Trp Gln Gly Ile His Phe Pro Arg 1 5 <210> 30 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 30 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Ile Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Val Arg Arg Gly Gly Tyr Phe Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 31 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 31 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaagcaggct 120 ccaggaaagg gtttaaagtg gatgggctgg ataaacacca acactggaga gccaacatat 180 gctgaagagt tcaagggacg gattgccttc tctttggaat cctctgccag cactgcctat 240 ttgcagatca acaacctcaa aaatgaggac acggccacat atttctgtgc aagagaggga 300 gtgcgacggg gggggtactt ttttgactac tggggccaag gcaccactct cacagtctcc 360 tca 363 <210> 32 <211> 109 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Ser Tyr His Cys Gln His Phe Trp Gly Phe Pro Phe 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 <210> 33 <211> 327 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 33 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtggggaga aactgtcacc 60 atcacatgtc gagcaagtga gaatatttac agcaatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct 240 gaagattttg ggagttatca ctgtcaacat ttttggggtt ttccattcac gttcggcgcg 300 gggacaaagt tggaaataaa acgggct 327 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 34 Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> 35 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 35 Asn Thr Asn Thr Gly Glu 1 5 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Glu Gly Val Arg Arg Gly Gly Tyr Phe Phe Asp Tyr 1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr 1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Glu Gly Val Arg Arg Gly Gly Tyr Phe Phe Asp Tyr 1 5 10 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Asn Tyr Gly Met Asn 1 5 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys 1 5 10 15 Gly <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Glu Gly Val Arg Arg Gly Gly Tyr Phe Phe Asp Tyr 1 5 10 <210> 43 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Thr Asn Tyr Gly Met Asn 1 5 <210> 44 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr 1 5 10 <210> 45 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 45 Ala Arg Glu Gly Val Arg Arg Gly Gly Tyr Phe Phe Asp 1 5 10 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 46 Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 47 Ala Ala Thr Asn Leu Ala Asp 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 48 Gln His Phe Trp Gly Phe Pro Phe Thr 1 5 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 49 Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 50 Ala Ala Thr Asn Leu Ala Asp 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 51 Gln His Phe Trp Gly Phe Pro Phe Thr 1 5 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 52 Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 53 Ala Ala Thr Asn Leu Ala Asp 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 54 Gln His Phe Trp Gly Phe Pro Phe Thr 1 5 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 55 Tyr Ser Asn Leu Ala Trp Tyr 1 5 <210> 56 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 56 Leu Leu Val Tyr Ala Ala Thr Asn Leu Ala 1 5 10 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 57 Gln His Phe Trp Gly Phe Pro Phe 1 5 <210> 58 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 58 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Ile Tyr Asp Gly Tyr Tyr Asp Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 59 <211> 357 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 59 gaggttcagc tgcagcagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg 60 tcctgcacag cttctggctt caacattaaa gacacctata tgcactgggt gaggcagagg 120 cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtta tactaaatat 180 gacccgaagt tccagggcaa ggccactata acagcagaca catcctccaa cacagcctac 240 ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtct catctatgat 300 ggttactacg actcctttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 60 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 60 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Pro Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Val 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 61 <211> 336 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 61 gatgttgtga tgacccagac tccactcact ttgtcggttc ccattggaca accagcctcc 60 atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattattgct ggcaagttac acattttcct 300 cggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 62 Gly Phe Asn Ile Lys Asp Thr 1 5 <210> 63 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 63 Asp Pro Ala Asn Gly Tyr 1 5 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 64 Tyr Asp Gly Tyr Tyr Asp Ser Phe Asp Tyr 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 65 Gly Phe Asn Ile Lys Asp Thr Tyr Met His 1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 66 Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys 1 5 10 <210> 67 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 67 Tyr Asp Gly Tyr Tyr Asp Ser Phe Asp Tyr 1 5 10 <210> 68 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 68 Asp Thr Tyr Met His 1 5 <210> 69 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 69 Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210> 70 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 70 Tyr Asp Gly Tyr Tyr Asp Ser Phe Asp Tyr 1 5 10 <210> 71 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 71 Lys Asp Thr Tyr Met His 1 5 <210> 72 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 72 Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys 1 5 10 <210> 73 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 73 Leu Ile Tyr Asp Gly Tyr Tyr Asp Ser Phe Asp 1 5 10 <210> 74 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 74 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 75 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 75 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 76 Trp Gln Val Thr His Phe Pro Arg Thr 1 5 <210> 77 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 77 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 78 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 78 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 79 Trp Gln Val Thr His Phe Pro Arg Thr 1 5 <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 80 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 81 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 81 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 82 Trp Gln Val Thr His Phe Pro Arg Thr 1 5 <210> 83 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 83 Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 84 Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp 1 5 10 <210> 85 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 85 Trp Gln Val Thr His Phe Pro Arg 1 5 <210> 86 <211> 128 <212> PRT <213> Homo sapiens <400> 86 Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His 1 5 10 15 Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr 20 25 30 Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr 35 40 45 Cys Val Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn 50 55 60 Ile Ser Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn 65 70 75 80 Gly Ser Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser 85 90 95 His Ser Thr Thr Leu Tyr Val Thr Gly Lys Gln Asn Glu Leu Ser Asp 100 105 110 Thr Ala Gly Arg Glu Ile Asn Leu Val Asp His His His His His His 115 120 125 <210> 87 <211> 11 <212> PRT <213> Homo sapiens <400> 87 Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys 1 5 10 <210> 88 <211> 11 <212> PRT <213> Homo sapiens <400> 88 Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys 1 5 10 <210> 89 <211> 12 <212> PRT <213> Homo sapiens <400> 89 Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg 1 5 10 <210> 90 <211> 12 <212> PRT <213> Homo sapiens <400> 90 Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg 1 5 10 <210> 91 <211> 11 <212> PRT <213> Homo sapiens <400> 91 Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg 1 5 10 <210> 92 <211> 32 <212> PRT <213> Homo sapiens <400> 92 Arg Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu 1 5 10 15 Cys Pro Val Lys Tyr Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys 20 25 30 <210> 93 <211> 20 <212> PRT <213> Homo sapiens <400> 93 Arg Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu 1 5 10 15 Cys Pro Val Lys 20 <210> 94 <211> 20 <212> PRT <213> Homo sapiens <400> 94 Arg Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu 1 5 10 15 Cys Pro Val Lys 20 <210> 95 <211> 19 <212> PRT <213> Homo sapiens <400> 95 Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys 1 5 10 15 Pro Val Lys <210> 96 <211> 19 <212> PRT <213> Homo sapiens <400> 96 Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys 1 5 10 15 Pro Val Lys <210> 97 <211> 31 <212> PRT <213> Homo sapiens <400> 97 Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys 1 5 10 15 Pro Val Lys Tyr Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys 20 25 30 <210> 98 <211> 12 <212> PRT <213> Homo sapiens <400> 98 Tyr Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys 1 5 10 <210> 99 <211> 21 <212> PRT <213> Homo sapiens <400> 99 Asn Ile Ser Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp 1 5 10 15 Asn Gly Ser Tyr Arg 20 <210> 100 <211> 22 <212> PRT <213> Homo sapiens <400> 100 Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser His Ser Thr Thr 1 5 10 15 Leu Tyr Val Thr Gly Lys 20 <210> 101 <211> 10 <212> PRT <213> Homo sapiens <400> 101 Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg 1 5 10 <210> 102 <211> 12 <212> PRT <213> Homo sapiens <400> 102 Glu Ile Asn Leu Val Asp His His His His His His 1 5 10 <210> 103 <211> 8 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (4)..(4) <223> Cys(carbamidomethyl) <400> 103 Lys Glu Ser Cys Asp Val Gln Leu 1 5 <210> 104 <211> 8 <212> PRT <213> Homo sapiens <400> 104 Lys Glu Ser Cys Asp Val Gln Leu 1 5 <210> 105 <211> 9 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (4)..(4) <223> Cys(carbamidomethyl) <400> 105 Lys Glu Ser Cys Asp Val Gln Leu Tyr 1 5 <210> 106 <211> 10 <212> PRT <213> Homo sapiens <400> 106 Ile Lys Arg Gln Ser Glu His Ser Ile Leu 1 5 10 <210> 107 <211> 15 <212> PRT <213> Homo sapiens <400> 107 Ile Lys Arg Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe 1 5 10 15 <210> 108 <211> 8 <212> PRT <213> Homo sapiens <400> 108 Glu Leu Glu Cys Pro Val Lys Tyr 1 5 <210> 109 <211> 17 <212> PRT <213> Homo sapiens <400> 109 Ile Lys Arg Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu 1 5 10 15 Leu <210> 110 <211> 13 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (9)..(9) <223> Cys(carbamidomethyl) <400> 110 Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr 1 5 10 <210> 111 <211> 9 <212> PRT <213> Homo sapiens <400> 111 Cys Ala Asn Arg Pro His Val Thr Trp 1 5 <210> 112 <211> 9 <212> PRT <213> Homo sapiens <400> 112 Lys Glu Ser Cys Asp Val Gln Leu Tyr 1 5 <210> 113 <211> 18 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (1)..(1) <223> Cys(carbamidomethyl) <220> <221> MOD_RES <222> (4)..(4) <223> Asn(Deamidated) <220> <221> MOD_RES <222> (8)..(8) <223> Cys(carbamidomethyl) <400> 113 Cys Lys Leu Asn Gly Thr Thr Cys Val Lys Leu Glu Asp Arg Gln Thr 1 5 10 15 Ser Trp <210> 114 <211> 18 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (1)..(1) <223> Cys(carbamidomethyl) <220> <221> MOD_RES <222> (8)..(8) <223> Cys(carbamidomethyl) <400> 114 Cys Lys Leu Asn Gly Thr Thr Cys Val Lys Leu Glu Asp Arg Gln Thr 1 5 10 15 Ser Trp <210> 115 <211> 15 <212> PRT <213> Homo sapiens <400> 115 Asn Gly Thr Cys Val Lys Leu Glu Asp Arg Gln Thr Ser Trp 1 5 10 15 <210> 116 <211> 9 <212> PRT <213> Homo sapiens <400> 116 Ile Glu Ser His Ser Thr Thr Leu Tyr 1 5 <210> 117 <211> 9 <212> PRT <213> Homo sapiens <400> 117 Lys Glu Glu Lys Asn Ile Ser Phe Phe 1 5 <210> 118 <211> 9 <212> PRT <213> Homo sapiens <400> 118 Lys Glu Ser Cys Asp Val Gln Leu Tyr 1 5 <210> 119 <211> 17 <212> PRT <213> Homo sapiens <400> 119 Glu Pro Val Leu Pro Asn Asp Asn Gly Ser Tyr Arg Cys Ser Ala Asn 1 5 10 15 Phe <210> 120 <211> 8 <212> PRT <213> Homo sapiens <400> 120 Val Thr Gly Lys Gln Asn Glu Leu 1 5 <210> 121 <211> 10 <212> PRT <213> Homo sapiens <400> 121 Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu 1 5 10 <210> 122 <211> 8 <212> PRT <213> Homo sapiens <400> 122 Asn Gly Thr Thr Cys Val Lys Leu 1 5 <210> 123 <211> 12 <212> PRT <213> Homo sapiens <400> 123 Gln Ser Asn Leu Ile Glu Ser His Ser Thr Thr Leu 1 5 10 <210> 124 <211> 8 <212> PRT <213> Homo sapiens <400> 124 Lys Glu Glu Lys Asn Ile Ser Phe 1 5 <210> 125 <211> 13 <212> PRT <213> Homo sapiens <400> 125 Gln Ser Asn Leu Ile Glu Ser His Ser Thr Thr Leu Tyr 1 5 10 <210> 126 <211> 9 <212> PRT <213> Homo sapiens <400> 126 Ile Glu Ser His Ser Thr Thr Leu Tyr 1 5 <210> 127 <211> 8 <212> PRT <213> Homo sapiens <400> 127 Glu Leu Glu Cys Pro Val Lys Tyr 1 5 <210> 128 <211> 18 <212> PRT <213> Homo sapiens <400> 128 Val Thr Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg Glu Ile 1 5 10 15 Asn Leu <210> 129 <211> 9 <212> PRT <213> Homo sapiens <400> 129 Cys Ala Asn Arg Pro His Val Thr Trp 1 5 <210> 130 <211> 6 <212> PRT <213> Homo sapiens <400> 130 Glu Cys Pro Val Lys Tyr 1 5 <210> 131 <211> 8 <212> PRT <213> Homo sapiens <400> 131 Glu Leu Glu Cys Pro Val Lys Tyr 1 5 <210> 132 <211> 8 <212> PRT <213> Homo sapiens <400> 132 Val Asp His His His His His His His 1 5 <210> 133 <211> 26 <212> PRT <213> Homo sapiens <400> 133 Val Thr Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg Glu Ile 1 5 10 15 Asn Leu Val Asp His His His His His His His 20 25 <210> 134 <211> 26 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (17)..(17) <223> Asn(Deamidated) <400> 134 Val Thr Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg Glu Ile 1 5 10 15 Asn Leu Val Asp His His His His His His 20 25 <210> 135 <211> 10 <212> PRT <213> Homo sapiens <400> 135 Ser Asp Thr Ala Gly Arg Glu Ile Asn Leu 1 5 10 <210> 136 <211> 9 <212> PRT <213> Homo sapiens <400> 136 Lys Glu Ser Cys Asp Val Gln Leu Tyr 1 5 <210> 137 <211> 18 <212> PRT <213> Homo sapiens <400> 137 Ser Asp Thr Ala Gly Arg Glu Ile Asn Leu Val Asp His His His His 1 5 10 15 His His <210> 138 <211> 7 <212> PRT <213> Homo sapiens <400> 138 Glu Asp Arg Gln Thr Ser Trp 1 5 <210> 139 <211> 8 <212> PRT <213> Homo sapiens <400> 139 Val Thr Gly Lys Gln Asn Glu Leu 1 5 <210> 140 <211> 9 <212> PRT <213> Homo sapiens <400> 140 Tyr Val Thr Gly Lys Gln Asn Glu Leu 1 5 <210> 141 <211> 17 <212> PRT <213> Homo sapiens <400> 141 Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile Leu Ala 1 5 10 15 Gly <210> 142 <211> 17 <212> PRT <213> Homo sapiens <400> 142 Asp Thr Ala Gly Arg Glu Ile Asn Leu Val Asp His His His His 1 5 10 15 His <210> 143 <211> 9 <212> PRT <213> Homo sapiens <400> 143 Lys Tyr Cys Ala Asn Arg Pro His Val 1 5 <210> 144 <211> 10 <212> PRT <213> Homo sapiens <400> 144 Asn Arg Pro His Val Thr Trp Cys Lys Leu 1 5 10 <210> 145 <211> 5 <212> PRT <213> Homo sapiens <400> 145 Lys Gln Asn Glu Leu 1 5 <210> 146 <211> 10 <212> PRT <213> Homo sapiens <400> 146 Leu Tyr Ile Lys Arg Gln Ser Glu His Ser 1 5 10 <210> 147 <211> 8 <212> PRT <213> Homo sapiens <400> 147 Leu Ile Glu Ser His Ser Thr Thr 1 5 <210> 148 <211> 8 <212> PRT <213> Homo sapiens <400> 148 Ile Lys Arg Gln Ser Glu His Ser 1 5 <210> 149 <211> 7 <212> PRT <213> Homo sapiens <400> 149 Lys Glu Ser Cys Asp Val Gln 1 5 <210> 150 <211> 7 <212> PRT <213> Homo sapiens <400> 150 Val Thr Gly Lys Gln Asn Glu 1 5 <210> 151 <211> 9 <212> PRT <213> Homo sapiens <400> 151 Ile Lys Arg Gln Ser Glu His Ser Ile 1 5 <210> 152 <211> 9 <212> PRT <213> Homo sapiens <400> 152 Val Lys Tyr Cys Ala Asn Arg Pro His 1 5 <210> 153 <211> 10 <212> PRT <213> Homo sapiens <400> 153 Ala Asn Arg Pro His Val Thr Trp Cys Lys 1 5 10 <210> 154 <211> 15 <212> PRT <213> Homo sapiens <400> 154 Val Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn 1 5 10 15 <210> 155 <211> 9 <212> PRT <213> Homo sapiens <400> 155 Ile Glu Ser His Ser Thr Thr Leu Tyr 1 5 <210> 156 <211> 9 <212> PRT <213> Mus sp. <400> 156 Ser Cys Ala Ala Ser Gly Phe Thr Phe 1 5 <210> 157 <211> 18 <212> PRT <213> Mus sp. <400> 157 Ser Val Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Leu 1 5 10 15 Ser Leu <210> 158 <211> 16 <212> PRT <213> Mus sp. <400> 158 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 1 5 10 15 <210> 159 <211> 14 <212> PRT <213> Mus sp. <400> 159 Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 1 5 10 <210> 160 <211> 9 <212> PRT <213> Mus sp. <400> 160 Ile Ser Cys Lys Ser Ser Leu Ser Leu 1 5 <210> 161 <211> 11 <212> PRT <213> Homo sapiens <400> 161 Tyr Ile Lys Arg Gln Ser Glu His Ser Ile Leu 1 5 10 <210> 162 <211> 7 <212> PRT <213> Homo sapiens <400> 162 Glu Asp Arg Gln Thr Ser Trp 1 5 <210> 163 <211> 10 <212> PRT <213> Homo sapiens <400> 163 Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu 1 5 10 <210> 164 <211> 8 <212> PRT <213> Homo sapiens <400> 164 Asn Gly Thr Thr Cys Val Lys Leu 1 5 <210> 165 <211> 11 <212> PRT <213> Homo sapiens <400> 165 Glu Pro Val Leu Pro Asn Asp Asn Gly Ser Tyr 1 5 10 <210> 166 <211> 11 <212> PRT <213> Homo sapiens <400> 166 Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile 1 5 10 <210> 167 <211> 11 <212> PRT <213> Homo sapiens <400> 167 Ile Lys Arg Gln Ser Glu His Ser Ile Leu Ala 1 5 10 <210> 168 <211> 11 <212> PRT <213> Homo sapiens <400> 168 Val Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys 1 5 10 <210> 169 <211> 11 <212> PRT <213> Homo sapiens <400> 169 Asn Gly Ser Tyr Arg Cys Ser Ala Asn Phe Gln 1 5 10

Claims (65)

Binds to B- and T-lymphocyte attenuator (BTLA),
(i)
(a) (1) a V _H complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 6;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 7; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 8
A heavy chain variable region (V _H ) comprising;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 18;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 19; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 20
As a light chain variable region (V _L ) comprising;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Chothia system;
or
(ii)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 9;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 10; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 11
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of an amino acid sequence of sequence number 21;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 22; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 23
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the AbM system;
or
(iii)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 12;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 13; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 14
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 24;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 25; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 26
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Kabat system;
or
(iv)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 15;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 16; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 17
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 27;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 28; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 29
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Contact system.
An antibody or antigen-binding fragment thereof comprising: In the first paragraph,
An antibody or antigen-binding fragment thereof comprising a V _H comprising the amino acid sequence of sequence number 2. In the first paragraph,
An antibody or antigen-binding fragment thereof comprising a V _L comprising the amino acid sequence of SEQ ID NO: 4. In the first paragraph,
An antibody or antigen-binding fragment thereof, comprising a V _H comprising the amino acid sequence of SEQ ID NO: 2 and a V _L comprising the amino acid sequence of SEQ ID NO: 4. Binds to B- and T-lymphocyte attenuating factor (BTLA),
(i)
(a) (1) a V _H complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 34;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 35; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 36
A heavy chain variable region (V _H ) comprising;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 46;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 47; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 48
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Chothia system;
or
(ii)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 37;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 38; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 39
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 49;
(2) V _L CDR2 consisting of an amino acid sequence of sequence number 50; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 51
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the AbM system;
or
(iii)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 40;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 41; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 42
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of an amino acid sequence of sequence number 52;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 53; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 54
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Kabat system;
or
(iv)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 43;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 44; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 45
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of an amino acid sequence of sequence number 55;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 56; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 57
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Contact system.
An antibody or antigen-binding fragment thereof comprising: In paragraph 5,
An antibody or antigen-binding fragment thereof comprising a V _H comprising the amino acid sequence of SEQ ID NO: 30. In paragraph 5,
An antibody or antigen-binding fragment thereof comprising a V _L comprising the amino acid sequence of SEQ ID NO: 32. In paragraph 5,
An antibody or antigen-binding fragment thereof, comprising a V _H comprising the amino acid sequence of SEQ ID NO: 30 and a V _L comprising the amino acid sequence of SEQ ID NO: 32. Binds to B- and T-lymphocyte attenuating factor (BTLA),
(i)
(a) (1) a V _H complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 62;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 63; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 64
A heavy chain variable region (V _H ) comprising;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 74;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 75; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 76
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Chothia system;
or
(ii)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 65;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 66; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 67
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of an amino acid sequence of sequence number 77;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 78; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 79
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the AbM system;
or
(iii)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 68;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 69; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 70
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of an amino acid sequence of sequence number 80;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 81; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 82
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Kabat system;
or
(iv)
(a) (1) a V _H CDR1 consisting of the amino acid sequence of sequence number 71;
(2) V _H CDR2 consisting of the amino acid sequence of sequence number 72; and
(3) V _H CDR3 consisting of the amino acid sequence of sequence number 73
V _H containing ;
and
(b) (1) a V _L CDR1 consisting of the amino acid sequence of sequence number 83;
(2) V _L CDR2 consisting of the amino acid sequence of sequence number 84; and
(3) V _L CDR3 consisting of the amino acid sequence of sequence number 85
As V _L including;
The above V _H CDR1-3 and V _L CDR1-3 are defined by the Contact system.
An antibody or antigen-binding fragment thereof comprising: In Article 9,
An antibody or antigen-binding fragment thereof comprising a V _H comprising the amino acid sequence of SEQ ID NO: 58. In Article 9,
An antibody or antigen-binding fragment thereof comprising a V _L comprising the amino acid sequence of SEQ ID NO: 60. In Article 9,
An antibody or antigen-binding fragment thereof, comprising a V _H comprising the amino acid sequence of SEQ ID NO: 58 and a V _L comprising the amino acid sequence of SEQ ID NO: 60. In any one of claims 1 to 12,
(i) Glycosylated BTLA compared to non-glycosylated BTLA;
(ii) BTLA glycosylated at position N75, N94, N110 or any combination thereof, compared to non-glycosylated BTLA;
(iii) a BTLA epitope comprising a sequence of five or more consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168 or 169; or
(iv) a BTLA epitope comprising at least one of the amino acids corresponding to R12, H16, K51, T57, S82 or S86 of BTLA of sequence number 86;
An antibody or antigen-binding fragment thereof that selectively binds to a specific antigen. In any one of claims 1 to 12,
An antibody or antigen-binding fragment thereof which specifically masks a glycosylation motif of BTLA comprising BTLA positions N75, N94, N110 or any combination thereof. In any one of claims 1 to 12,
An antibody or antigen-binding fragment thereof, wherein binding of the antibody or antigen-binding fragment thereof to glycosylated BTLA is indicated in a fluorescence assay by a fluorescence intensity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold greater than the fluorescence intensity shown to non-glycosylated BTLA. In any one of claims 1 to 12,
(i) the antibody inhibits binding of herpesvirus-entry mediator (HVEM) to BTLA;
(ii) the antibody is recombinant;
(iii) the antibody is IgG, IgM, IgA or an antigen-binding fragment thereof;
(iv) the antibody is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv or single domain antibody;
(v) the antibody is a human or humanized antibody; or
(vi) the antibody is conjugated to an imaging agent, chemotherapeutic agent, toxin or radionuclide;
An antibody or an antigen-binding fragment thereof. A composition for treating cancer in a subject in need of cancer treatment, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising an antibody or an antigen-binding fragment thereof of any one of claims 1 to 12. In Article 18,
A pharmaceutical composition, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer or rectal cancer. In Article 19,
A pharmaceutical composition for use in combination with at least a second anticancer treatment. A pharmaceutical composition in claim 20, wherein the second anticancer treatment is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. A method for assessing BTLA glycation, comprising contacting an antibody or antigen-binding fragment thereof of any one of claims 1 to 12 with a BTLA-containing sample. A method for producing an antibody according to any one of claims 1 to 4, comprising administering to a non-human animal a composition comprising a polypeptide and a pharmaceutically acceptable carrier and isolating the antibody from the non-human animal,
The above polypeptide
(a) at least seven consecutive amino acids of human BTLA comprising at least one amino acid corresponding to positions N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to positions N75, N94, and N110 of human BTLA is glycosylated;
(b) a sequence of five or more consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168 or 169; or
(c) an amino acid corresponding to R12, H16, K51, T57, S82 or S86 of BTLA of sequence number 86;
A method for producing an antibody according to any one of claims 1 to 4, comprising: A method for producing an antibody according to any one of claims 1 to 4, comprising administering a polypeptide to a non-human animal and isolating an antibody from the non-human animal,
The above polypeptide
(a) at least seven consecutive amino acids of human BTLA comprising at least one amino acid corresponding to positions N75, N94, or N110 of human BTLA, wherein at least one of said amino acids corresponding to positions N75, N94, and N110 of human BTLA is glycosylated;
(b) a sequence of five or more consecutive amino acids of the amino acid sequence of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168 or 169; or
(c) an amino acid corresponding to R12, H16, K51, T57, S82 or S86 of BTLA of sequence number 86;
A method for producing an antibody according to any one of claims 1 to 4, comprising: delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete